Methods for producing microRNAs

ABSTRACT

The invention relates to recombinant vectors for inducible and/or tissue specific expression of double-stranded RNA molecules that interfere with the expression of a target gene. In certain embodiments, the invention relates to the use of Tet (tetracycline)-responsive RNA Polymerase II (Pol II) promoters (e.g., TetON or TetOFF) to direct inducible knockdown in certain cells of an integrated or an endogenous gene, such as p53. The invention also relates to a method for producing transgenic animals (e.g., mice) expressing inducible (such as tetracycline-regulated), reversible, and/or tissue-specific double-stranded RNA molecules that interfere with the expression of a target gene.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 11/444,107, filed onMay 31, 2006, which claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/686,135, entitled “METHODS FORPRODUCING MICRORNAS,” filed on May 31, 2005. The entire teachings of theabove-referenced applications are incorporated herein by reference.

GOVERNMENT SUPPORT

Work described herein was funded, in whole or in part, by Mouse Modelsof Human Cancer Consortium Grant No. 25480211. The United Statesgovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) has been used to silence the expression of atarget gene. RNAi is a sequence-specific post-transcriptional genesilencing mechanism triggered by double-stranded RNA (dsRNA). It causesdegradation of mRNAs homologous in sequence to the dsRNA. The mediatorsof the degradation are 21-23-nucleotide small interfering RNAs (siRNAs)generated by cleavage of longer dsRNAs (including hairpin RNAs) byDICER, a ribonuclease III-like protein. Molecules of siRNA typicallyhave 2-3-nucleotide 3′ overhanging ends resembling the RNAse IIIprocessing products of long dsRNAs that normally initiate RNAi. Whenintroduced into a cell, they assemble an endonuclease complex(RNA-induced silencing complex), which then guides target mRNA cleavage.As a consequence of degradation of the targeted mRNA, cells with aspecific phenotype of the suppression of the corresponding proteinproduct are obtained (e.g., reduction of tumor size, metastasis,angiogenesis, and growth rates).

The small size of siRNAs, compared with traditional antisense molecules,prevents activation of the dsRNA-inducible interferon system present inmammalian cells. This helps avoid the nonspecific phenotypes normallyproduced by dsRNA larger than 30 base pairs in somatic cells. See, e.g.,Elbashir et al., Methods 26:199-213 (2002); McManus and Sharp, NatureReviews 3:737-747 (2002); Hannon, Nature 418:244-251 (2002); Brummelkampet al., Science 296:550-553 (2002); Tuschl, Nature Biotechnology20:446-448 (2002); U.S. Application US2002/0086356

SUMMARY OF THE INVENTION

One aspect of the invention provides an artificial nucleic acidconstruct comprising an RNA Polymerase II (Pol II) promoter operablylinked to a coding sequence for expressing a precursor molecule for ansiRNA, the siRNA inhibiting the expression of a target gene, wherein thenucleic acid construct directs the expression of the precursor moleculeand/or the siRNA, and substantially inhibits the expression of thetarget gene when the artificial nucleic acid construct is stablyintegrated into a host cell genome.

In certain embodiments, the Pol II promoter is an inducible promoter, atissue-specific promoter, or a developmental stage-specific promoter.For example, the inducible promoter may be a tetracyclin-responsivepromoter, including commercially available TetON promoter (thetranscription from which promoter is activated at the presence oftetracyclin (tet), doxycycline (Dox), or tet analog), or the TetOFFpromoter (the transcription from which promoter is turned off at thepresence of tetracyclin (tet), doxycycline (Dox), or a tet analog),e.g., those from Clontech, Inc.

In other embodiments, the inducible promoter may be selected from: apromoter operably linked to a lac operator (LacO), a LoxP-stop-LoxPsystem promoter, or a GeneSwitch™ or T-REx™ system promoter(Invitrogen), or equivalents thereof with identical or substantiallysimilar mechanisms.

In yet other embodiments, the Pol II promoter can be any art-recognizedPol II promoters, such as an LTR promoter or a CMV promoter.

In certain embodiments, the precursor molecule may be a precursormicroRNA, such as an artificial miR comprising coding sequence for thesiRNA for the target gene. For example, the miR may comprise a backbonedesign of microRNA-30 (miR-30). Alternatively, the miR may comprise abackbone design of miR-15a, -16, -19b, -20, -23a, -27b, -29a, -30b,-30c, -104, -132s, -181, -191, -223. See US 2005/0075492A1 (incorporatedherein by reference).

In other embodiments, the precursor molecule may be a short hairpin RNA(shRNA).

The constructs of the instant invention is highly potent, and a singleintegrated copy of the subject nucleic acid construct is sufficient forsubstantially inhibiting the expression of the target gene.“Substantially inhibiting” as used herein includes inhibiting at leastabout 20%, or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or closeto 100% of the expression or mRNA and/or protein of the target gene.

The constructs of the invention may further comprise an enhancer for thePol II promoter.

The constructs of the invention may further comprise a reporter geneunder the control of a second promoter, such as a luciferase, afluorescent protein (e.g. GFP, RFP, YFP, BFP, etc.), or an enzyme, orany other art-recognized reporter whose physical presence and/oractivity can be readily assessed using an art-recognized method.

In certain embodiments, the second promoter and the reporter gene can bedownstream of (3′-to) the coding sequence for the precursor molecule. Inother embodiments, the reporter gene is translated from an internalribosomal entry site (IRES) between a second promoter and the reportergene.

In other embodiments, the coding sequence for expressing the precursormolecule may be embedded or inserted into the 5′-UTR (5′-untranslatedregion), 3′-UTR, or an intron of the reporter gene.

The constructs of the invention may further comprise at least oneselectable marker, such as puromycin, zeocin, hygromycin, or neomycin,etc.

The constructs of the invention may further comprise a Pol III promoterupstream of the coding sequence for expressing the precursor molecule.

The constructs of the invention can be used to inhibit the expression ofa number of different target genes. In certain embodiments, the targetgene is associated with a disease condition such as cancer or infectiousdisease. For example, the target gene may be over-expressed orabnormally active in the disease. In addition, the target gene may be anoncogene or an antagonist/inhibitor or dominant negative mutation of atumor suppressor gene.

Another aspect of the invention provides a cell comprising any of thesubject nucleic acid constructs.

In certain embodiments, the cell may be a mammalian cell.

In certain embodiments, the cell may be a tissue culture cell (e.g., aprimary cell, or a cell from an established cell line), a cell in vivo,or a cell manipulated ex vivo.

If the Pol II promoter is an inducible promoter, the cell may furthercomprise an additional construct for expressing an activator or aninhibitor of the inducible promoter. For example, if the induciblepromoter is a tet-responsive promoter, the additional construct mayencode tTA or rtTA. If the inducible promoter is a LacO-responsivepromoter, the additional construct may encode LacI. If the induciblepromoter is a LoxP-stop-LoxP system promoter, the additional constructmay encode a Cre recombinase, which may be under the transcriptionalcontrol of an inducible promoter, a developmental stage-specificpromoter, or a tissue-specific promoter.

Another aspect of the invention provides a non-human mammal comprisingany of the subject cells described above. In certain embodiments, thenon-human mammal may be a chimeric mammal some of whose somatic or germcells are subject cells as described above. Alternatively, the non-humanmammal may be a transgenic mammal all of whose somatic or germ cells aresubject cells described above.

Another aspect of the invention provides a method for making a subjectchimeric non-human mammal as described above, comprising introducing aconstruct according to any of the subject nucleic acid constructs intoan embryonic stem (ES) cell and generating a chimeric mammal from the EScell.

Another aspect of the invention provides a method for making a subjecttransgenic non-human mammal described above, comprising mating a subjectchimeric non-human mammal described above with another animal from thesame species.

Another aspect of the invention provides a method for inhibiting theexpression of a target gene of interest in a cell, comprisingintroducing a subject construct into the cell, wherein the siRNAmolecule derived from the precursor molecule is specific for the targetgene.

In certain embodiments, the method further comprises inhibiting at leastone additional target gene(s) of interest in the cell by introducing atleast one additional constructs according to any one of the subjectnucleic acid constructs into the cell, wherein each of the siRNAmolecules derived from the precursor molecules are specific for theadditional target genes, respectively.

Another aspect of the invention provides a method for treating agene-mediated disease, comprising introducing into an individual havingthe disease a construct according to any of the subject nucleic acidconstructs, where the siRNA derived from the precursor molecule isspecific for the gene mediating the disease.

Another aspect of the invention provides a method of validating a geneas a potential target for treating a disease, comprising: (1)introducing a construct according to any one of the subject nucleic acidconstructs described herein into a cell associated with the disease,wherein the siRNA molecule derived from the precursor molecule isspecific for the gene; (2) assessing the effect of inhibiting theexpression of the gene on one or more disease-associated phenotype;wherein a positive effect on at least one disease-associated phenotypeis indicative that the gene is a potential target for treating thedisease.

In certain embodiments, the gene is over-expressed or abnormally activein disease cells or tissues. Alternatively, the gene may be downstreamof and is activated by a second gene over-expressed or abnormally activein disease cells or tissues. In addition, the product of the geneantagonizes an suppressor of a second gene over-expressed or abnormallyactive in disease cells or tissues.

In certain embodiments, the cell may be a tissue culture cell, such as aprimary cell isolated from diseased tissues, or from an established cellline derived from diseased tissues.

In other embodiments, the cell is within diseased tissues, and step (2)above comprises evaluating one or more symptoms of the disease.

In certain embodiments, the cell may be one from a transgenic animal,such as one comprising any of the subject nucleic acid constructs.

For example, in a transgenic animal with a transgene comprising any ofthe subject nucleic acid constructs, the transgene may encode aprecursor molecule, which, upon processing, generates a siRNA specificfor the candidate target gene. Preferably, the expression of theprecursor molecule is inducible, reversible, and/or tissue-specific.

In certain embodiments, the method further comprises assessing the sideeffect, if any, of knocking down the expression of the target gene inone or more tissues/organs other than the diseased tissue, wherein thetarget gene is a valid target if the side effect, if any, is acceptableto a person of skill in the respective art (e.g., when validating a drugtarget, such side effects resulting from impairment of the target genefunction in other tissues must be acceptable to a physician orveterinarian).

In certain embodiments, the expression of the gene may be induciblyinhibited by a subject construct, or inducibly activated by turning downthe expression of a subject construct.

It is also contemplated that all embodiments of the invention, includingthose specifically described for different aspects of the invention, canbe combined with any other embodiments of the invention as appropriate.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows effective knockdown via single copy expression ofmiR30-based shRNAs from a retroviral LTR promoter. (A) Schematicrepresentation of predicted RNA folds for simple stem/loop and miR30design shRNAs. Note extensive predicted folding for the ˜300 ntpre-miR30 RNA. Folds were generated using mfold. (B) Retroviral vectorsused to deliver shRNAs to mammalian cells. Provirus layouts are shown toindicate promoter activity of the integrated virus. Active promoters areshown as open arrows, with two inverted black arrows representing shRNAstem sequences. (C) Western blot analysis for p53 expression of NIH3T3cells transduced with the retroviral vectors shown in B and selected inpuromycin. A tubulin blot is shown as a loading control. (D) Colonyformation assay for the cells shown in (C). Cells were seeded in 6 wellplates at 2500 cells/well, and allowed to grow for 10 days beforeharvesting. (E) Western blot analysis for p53 expression in NIH3T3 cellstransduced at less than 5% efficiency (assessed by GFP FACS; not shown)with the retroviral vectors shown in (B). A tubulin blot is shown as aloading control. Similar results were obtained in other cell typesincluding wild type and p19ARF-null MEFs (data not shown).

FIG. 2 shows that RNA polymerase II-driven shRNAs can effectivelypromote tumorigenesis and chemotherapy resistance in vivo. (A)Kaplan-Meier curve showing mouse survival following adoptive transfer ofEμ-Myc HSCs infected with LTR-driven Bim shRNAs. (B) Western blotshowing reduced BimEL and BimL expression in Eμ-Myc lymphomas expressingBim shRNAs. Control: archived tumors arising from Eμ-Myc HSCs (on eithera wild type, ARF^(+/−) or p53^(+/−) background; not shown) were used ascontrols for Bim expression. (C) Kaplan-Meier curves showing tumor-freesurvival (left) and overall survival (right) for mice harboringp19ARF-null lymphomas infected with either the LMP-p53. 1224 retrovirusor vector control. Tumor-bearing mice were given a single 10 mg/kg doseof adriamycin at day zero. (D) Flow cytometry analysis of GFP expressionin lymphoma cells harvested from the mice in (A). Representativehistograms show the percent of GFP-positive cells at the time oftreatment (left) and after tumor relapse (right).

FIG. 3 shows stable and regulatable shRNA expression from atet-responsive RNA polymerase II promoter. (A) Provirus layout of theSIN-TREmiR30-PIG (TMP) retroviral vector. (B) Western blot analysis ofRb expression in HeLa-tTA cells infected with TMP-Rb.670. Cells weretreated with 100 ng/mL Dox for 4 days prior to harvesting. Controluninfected HeLa-tTA cells treated with Dox are also shown. (C) Rbexpression in homogeneous cultures derived from single-cell clones ofHeLa-tTA cells infected at single copy with TMP-Rb.670. Cells werecultured in normal, Dox-free medium prior to harvesting. con=controluninfected HeLa-tTA cells. (D) Dox dose/response analysis of Rbexpression in HeLa-tTA clone Rb.670C. Cells were cultured for 8 days inthe indicated Dox concentration prior to harvesting. Control uninfectedHeLa-tTA cells (con) cultured with or without Dox are also shown. Notethe presence of a non-specific band in the GFP immunoblot, running justbelow GFP. (E) Rb expression in HeLa-tTA clone Rb.670C cells over timein response to shifting into or out of Dox. Cells were cultured withoutDox (left panels) or in 100 ng/mL Dox (right panels) for eight daysprior to shifting them into 100 ng/mL Dox or Dox-free medium,respectively. Again, note the presence of a faint non-specific band inthe GFP immunoblot. Similar results were observed for all Rb.670 clonesshowing good Rb knockdown in Dox-free medium (C, above), with someclonal variation in kinetics. In addition, similar results were obtainedusing other mishRNAs targeting Rb and PTEN (data not shown). (F) Rbexpression in homogeneous cultures derived from single-cell clones ofU2OS-rtTA cells infected at around 1% efficiency with TMP-Rb.670. Cellswere cultured in 1000 ng/mL Dox for several days prior to harvesting.(G) Dox dose/response analysis of Rb expression in U2OS-rtTA cloneRb.670R5 cells. Cells were cultured for 8 days in the indicated Doxconcentration prior to harvesting. Control uninfected U2OS-rtTA cellscultured with or without Dox are also shown. (H) Rb expression inU2OS-rtTA clone Rb.670R5 cells in response to Dox treatment. Cells werecultured without Dox for eight days prior to shifting them into 1000ng/mL Dox. Note that Rb.670R5 cells express some GFP in Dox-free medium,and Rb levels are slightly decreased compared with controls, indicatingslightly leaky expression from the TRE-CMV promoter in this particularclone.

FIG. 4 shows reversible p53 knockdown in primary MEFs. (A) Colonyformation assays of wild type MEFs doubly infected with TMP-p53.1224 andtTA. Cells were seeded in 6 well plates at 5000 cells/well, and grownfor 8 days before harvesting. Upper wells contained Dox-free medium,whereas lower wells contained 100 ng/mL Dox. Positive control p53-nullMEFs are shown, as are negative control wild type MEFs infected withTMP-p53. 1224 alone or with TMP-PTEN.1010 plus tTA. (B) Western blotanalysis of p53 and GFP expression in cells expanded from a single-cellclone of wild type MEFs infected with TMP-p53. 1224 and tTA (WtT cells).Cells were cultured in 100 ng/mL Dox for various times prior toharvesting. (C) Morphology and GFP fluorescence of WtT cells originallyplated at colony formation density, and cultured in Dox-free medium(upper panels) or 100 ng/mL Dox (lower panels). Right panel: SA-β-galstaining of WtT cells cultured in Dox-free medium (upper) or 100 ng/mLDox (lower). (D) Left panel: Colony formation assay for WtT cellscultured for 8 days in 100 ng/mL Dox, then seeded in Dox free medium(upper well) or 100 ng/mL Dox (lower well). Right panel: Colonyformation assay of cells equivalent to those in the upper well of theleft panel (formerly Dox-treated, dormant WtT cells after extendedculture in Dox-free medium). Cells were seeded and harvested as in (A).(E) Morphology, GFP fluorescence, and SA-β-gal staining of WtT cellsinfected with Ras and cultured in normal medium (upper panels) or 100ng/mL Dox (lower panels). (F) Western blot analysis of p53 and GFPexpression in WtT cells infected with Ras. Cells were cultured in 100ng/mL Dox for various times prior to harvesting.

FIG. 5 shows regulated p53 knockdown in tumors. (A) GFP and standardimaging of representative tumor-bearing nude mice, with Dox treatmentcommencing at day 0 (lower panels). Untreated controls are shown (upperpanels). (B) Representative tumor growth curves for WtT-Ras tumors in anuntreated mouse (open squares), or a mouse treated for 10 days with Dox(filled circles indicate Dox treatment) commencing at day 0. Each datapoint is the average volume of 2 tumors for a single mouse. Similarresults were obtained for 8 different WtT-Ras clones, with slightlydiffering kinetics. (C) Representative tumor growth curves forWtT-E1A/Ras tumors in an untreated mouse (open squares), or a mousetreated for 7 days with Dox (filled circles indicate Dox treatment)commencing at day 0. Each data point is the average volume of 2 tumorsfor a single mouse. Insets show GFP status of a single tumor at varioustimes. (D) Histological analysis of cell morphology and apoptosis inrepresentative nude mouse tumors harvested from untreated mice or micetreated with Dox for several days. (E) Western blot analysis of p53 andGFP expression in representative WtT-Ras and WtT-E1 A/Ras tumorsharvested from untreated mice or mice treated with Dox for several days.Cultured WtT-Ras cells treated with Dox are shown as a control.

FIG. 6 shows an siRNA northern blot of tissues isolated from animals ofvarious genotypes, probed with a labelled oligonucleotide thathybridizes to the p53.1224 siRNA. The individual lanes are: M: Molecularweight marker; 1: LAP-tTA liver; 2: LAP-tTA;TRE-1224 liver; 3:LAP-tTA;TRE-1224 liver after 4 days Doxycycline administration; 4:LAP-tTA spleen; 5: LAP-tTA;TRE-1224 spleen; 6: LAP-tTA;TRE-1224 spleenafter 4 days Doxycycline administration; 7: Eu-myc;TRE-1224 mouse 4-1spleen; 8: Eu-myc;TRE-1224 mouse 6-4 spleen; 9: Eu-myc;Eu-tTA;TRE-1224mouse #1 spleen; 10: Eu-myc;Eu-tTA;TRE-1224 mouse #2 spleen; 11:Eu-myc;Eu-tTA;TRE-1224 mouse #1-2 spleen; 12 & 13: Spleen (12) and lymphnode (13) from a tumor-bearing nude mouse recipient of Eu-myc lymphomacells; 14 & 15: Spleen (14) and lymph node (15) from a tumor-bearingnude mouse recipient of Eu-myc;Eu-tTA;TRE-1224 lymphoma cells; 16 & 17:Spleen (16) and lymph node (17) from a tumor-bearing nude mouserecipient of Eu-myc;Eu-tTA;TRE-1224 lymphoma cells, after 14 daysDoxycycline administration.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

RNA interference (RNAi) is normally triggered by double stranded RNA(dsRNA) or endogenous microRNA precursors (pre-miRNAs). Since itsdiscovery, RNAi has emerged as a powerful genetic tool for suppressinggene expression in mammalian cells. Stable gene knockdown can beachieved by expression of synthetic short hairpin RNAs (shRNAs),traditionally from RNA polymerase III promoters.

The instant invention generally relates to the use of RNA Polymerase IIpromoters to express microRNA (miRNA) precursors and/or short hairpinRNAs (shRNAs), either in vitro, ex vivo, or in vivo, especially from asfew as one single stably integrated expression construct. The singleexpression construct may be stably transfected/infected into a targetcell, or may be a germline transgene. Transgenic animals with thesubject RNAi constructs, which may be regulated to express mishRNA in aninducible, reversible, and/or tissue-specific manner, can be used toestablish valuable animal models for certain disease, such as thoseassociated with loss-of-function of certain target genes. The ability tocontrol both the timing (e.g., at certain developmental stages) andlocation (e.g., tissue-specific) of target gene knock-down, includingthe ability to reverse the course of induction/inactivation, renders thesubject system a powerful tool to study gene function and diseaseprogression. Such animal models or cells thereof may also be used fordrug screening or validation.

In certain embodiments, Pol II promoters controls the transcription ofthe subject miRNA/shRNA coding sequence. In general, any Pol IIcompatible promoters may be used for the instant invention.

In certain embodiments, various inducible Pol II promoters may be usedto direct precursor miRNA/shRNA expression. Exemplary inducible Pol IIpromoters include the tightly regulatable Tet system (either TetOn orTetOFF), and a number of other inducible expression systems known in theart and/or described herein. The tet systems allows incremental andreversible induction of precursor miRNA/shRNA expression in vitro and invivo, with no or minimal leakiness in precursor miRNA/shRNA expression.Such inducible system is advantages over the existing unidirectionalCre-lox strategies. Other systems of inducible expression may also beused with the instant constructs and methods.

In certain embodiments, expression of the subject miRNA/shRNA may beunder the control of a tissue specific promoter, such as a promoter thatis specific for: liver, pancreas (exocrine or endocrine portions),spleen, esophagus, stomach, large or small intestine, colon, GI tract,heart, lung, kidney, thymus, parathyroid, pineal gland, pituitory gland,mammary gland, salivary gland, ovary, uterus, cervix (e.g., neckportion), prostate, testis, germ cell, ear, eye, brain, retina,cerebellum, cerebrum, PNS or CNS, placenta, adrenal cortex or medulla,skin, lymph node, muscle, fat, bone, cartilage, synovium, bone marrow,epithelial, endothelial, vescular, nervous tissues, etc. The tissuespecific promoter may also be specific for certain disease tissues, suchas cancers. See Fukazawa et al., Cancer Research 64: 363-369, 2004(incorporated herein by reference).

Any tissue specific promoters may be used in the instant invention.Merely to illustrate, Chen et al. (Nucleic Acid Research, Vol. 34,database issue, pages D104-D107, 2006) described TiProD, theTissue-specific Promoter Database (incorporated herein by reference).Specifically, TiProD is a database of human promoter sequences for whichsome functional features are known. It allows a user to query individualpromoters and the expression pattern they mediate, gene expressionsignatures of individual tissues, and to retrieve sets of promotersaccording to their tissue-specific activity or according to individualGene Ontology terms the corresponding genes are assigned to. Thedatabase have defined a measure for tissue-specificity that allows theuser to discriminate between ubiquitously and specifically expressedgenes. The database is accessible at tiprod.cbi.pku dotedu.cn:8080/index.html. It covers most (if not all) the tissuesdescribed above.

In certain embodiments, if the reversibly inducible systems of theinvention are used, the subject shRNAs are not designed to target thepromoter regions of a target gene to avoid irreversible TGS.

In certain embodiments, artificial miRNA constructs based on, forexample, miR30 (microRNA 30), may be used to express precursormiRNA/shRNA from single/low copy stable integration in cells in vivo, orthrough germline transmission in transgenic animals. For example, Silvaet al. (Nature Genetics 37: 1281-88, 2005, incorporated herein byreference) have described extensive libraries of pri-miR-30-basedretroviral expression vectors that can be used to down-regulate almostall known human (at least 28,000) and mouse (at least 25,000) genes (seeRNAi Codex, a single database that curates publicly available RNAiresources, and provides the most complete access to this growingresource, allowing investigators to see not only released clones butalso those that are soon to be released, available at http://codex.cshldot edu). Although such libraries are driven by Pol III promoters, theycan be easily converted to the subject Pol II-driven promoters (seeMethods in Dickins et al., Nat. Genetics 37: 1289-95, 2005; also seepage 1284 in Silva et al., Nat. Genetics 37: 1281-89, 2005).

In certain embodiments, even a single copy of stably integratedprecursor miRNA/shRNA construct results in effective knockdown of atarget gene.

In certain embodiments, the inducible Tet system, coupled with thelow-copy integration feature of invention, allows more flexiblescreening applications, such as in screening for potentially lethalshRNAs or synthetic lethal shRNAs.

In certain embodiments, the subject precursor miRNA cassette may beinserted within a gene encoded by the subject vector. For example, thesubject precursor miRNA coding sequence may be inserted with an intron,the 5′- or 3′-UTR of a reporter gene such as GFP, etc.

In certain embodiments, cultured cells, such as wild type mousefibroblasts or primary cells can be switched from proliferative tosenescent states simply through regulated knockdown of p53 using thesubject constructs and methods.

The constructs and methods of the invention is advantageous in severalrespects.

In one respect, stable precursor miRNA/shRNA expression may be effectedthrough retroviral or lentiviral delivery of the miRNA/shRNAs, which isshown to be effective at single copy per cell. This allows veryeffective stable gene expression regulation at extremely low copy numberper cell (e.g. one per cell), thus vastly advantageous over systemsrequiring the introduction of a large copy number of constructs into thetarget cell by, for example, transient transfection.

Compare to transfection where there are multiple copies (such asmultiple episomal copies) of the shRNA construct, and the LTR is active,the instant system is preferable for stable expression of the shRNA.

Using the instant system, Applicants have discovered rapid andcoordinated entry into senescence upon re-establishment of wild type p53expression in p53 defective cells. Such an observation would not havebeen possible using previous technologies.

Another useful feature of the invention is that it is compatible with anestablished miR30 miRNA/shRNA library, which contains designedmiRNA/shRNA constructs targeting almost all human and mouse genes. Anyspecific member of the library can be readily cloned (such as by PCR)into the vectors of the instant invention for Pol II-driven regulatedand stable expression.

Other vector designs with different promoters have shown dependence onposition of transcriptional start and stop sites. The subjectmethod/system apparently has no such stringent requirements.

Applicants have also discovered that promoter interference between PolII and Pol III promoters may prevent efficient transcription of encodedshRNA, while the use of miRNA precursor has largely overcome thisproblem.

Another aspect of the invention provides a method for drug targetvalidation. The outcome of inhibiting the function of a gene, especiallythe associated effect in vivo, is usually hard to predict. Geneknock-out experiments offer valuable data for this purpose, but isexpensive, time consuming, and potentially non-informative since manygenes are required for normal development, such that loss-of-functionmutation in such genes causes embryonic lethality. Using the methods ofthe instant invention, especially the inducible expression regulationsystem of the invention, any potential drug target/candidate gene fortherapeutic intervention may be tested first by selectively up- and/ordown-regulating their expression in vitro, ex vivo, or in vivo, anddetermining the effect of such regulated expression, especially in vivoeffects on an organism. If disruption of the normal expression patternof a candidate gene shows desired phenotypes in vitro and/or in vivo,the candidate gene is chosen as a target for therapeutic intervention.Various candidate compounds can then be screened to identify inhibitorsor activators of such validated targets.

Another aspect of the invention provides a method to determine theeffect of coordinated expression regulation of two or more genes. Forexample, miRNA/shRNA constructs for two more target genes may beintroduced into a target cell (e.g., by stable integration) or anorganism (e.g., by viral vector infection or transgenic techniques), andtheir expression may be individually or coordinately regulated using theinducible and/or tissue specific or developmental specific promotersaccording to the instant invention. Since different inducible promotersare available, the expression of the two or more target genes may beregulated either in the same or opposite direction (e.g., both up- ordown-regulating, or one up one down, etc.). Such experiments can provideuseful information regarding, inter alia, genetic interaction betweenrelated genes.

In certain embodiments, the instant invention allows highly efficientknockdown of a target gene from a single (retroviral) integration event,thus providing a highly efficient means for certain screeningapplications. For example, the instant system and methods may be used totest potentially lethal miRNA/shRNAs or synthetic lethal miRNA/shRNAs.

The invention also provide a method to treat certain cancer, especiallythose cancer overexpressing Ras pathway genes (e.g., Ras itself) andhaving impaired p53 function, comprising introducing into such cells anactive p53 gene or gene product to induce senescence and/or apoptosis,thereby killing the cancer cells, or at least inhibit cancer progressionand/or growth.

The general feature of the invention having been described, thefollowing section provides certain illustrative aspects of the inventionthat may be combined in specific embodiments. Other similar orequivalent art-recognized methods may also be readily adapted for use inthe instant invention.

II. MicroRNA and RNAi Design

DNA vectors that express perfect complementary short hairpins RNAs(shRNAs) are commonly used to generate functional siRNAs. However, theefficacy of gene silencing mediated by different short-hairpin derivedsiRNAs may be inconsistent, and a substantial number of short-hairpinsiRNA expression vectors can trigger an anti-viral interferon response(Nature Genetics 34: 263, 2003). Moreover, siRNA short-hairpins aretypically processed symmetrically, in that both the functional siRNAstrand and its complement strand are incorporated into the RISC complex.Entry of both strands into the RISC can decrease the efficiency of thedesired regulation and increase the number of off-target mRNAs that areinfluenced. In comparison, endogenous microRNA (miRNA) processing andmaturation is a fairly efficient process that is not expected to triggeran anti-viral interferon response. This process involves sequentialsteps that are specified by the information contained in miRNA hairpinand its flanking sequences.

MicroRNAs (miRNAs) are endogenously encoded ˜22-nt-long RNAs that aregenerally expressed in a highly tissue- or developmental-stage-specificfashion and that post-transcriptionally regulate target genes. More than200 distinct miRNAs having been identified in plants and animals, thesesmall regulatory RNAs are believed to serve important biologicalfunctions by two prevailing modes of action: (1) by repressing thetranslation of target mRNAs, and (2) through RNA interference (RNAi),that is, cleavage and degradation of mRNAs. In the latter case, miRNAsfunction analogously to small interfering RNAs (siRNAs). Importantly,miRNAs are expressed in a highly tissue-specific or developmentallyregulated manner and this regulation is likely key to their predictedroles in eukaryotic development and differentiation. Analysis of thenormal role of miRNAs will be facilitated by techniques that allow theregulated over-expression or inappropriate expression of authenticmiRNAs in vivo, whereas the ability to regulate the expression of siRNAswill greatly increase their utility both in cultured cells and in vivo.Thus one can design and express artificial microRNAs based on thefeatures of existing microRNA genes, such as the gene encoding the humanmiR-30 microRNA. These miR30-based shRNAs have complex folds, and,compared with simpler stem/loop style shRNAs, are more potent atinhibiting gene expression in transient assays.

miRNAs are first transcribed as part of a long, largely single-strandedprimary transcript (Lee et al., EMBO J. 21: 4663-4670, 2002). Thisprimary miRNA transcript is generally, and possibly invariably,synthesized by RNA polymerase II (pol II) and therefore is normallypolyadenylated and may be spliced. It contains an ˜80-nt hairpinstructure that encodes the mature ˜22-nt miRNA as part of one arm of thestem. In animal cells, this primary transcript is cleaved by a nuclearRNaseIII-type enzyme called Drosha (Lee et al., Nature 425: 415-419,2003) to liberate a hairpin miRNA precursor, or pre-miRNA, of ˜65 nt,which is then exported to the cytoplasm by exportin-5 and the GTP-boundform of the Ran cofactor (Yi et al., Genes Dev. 17: 3011-3016, 2003).Once in the cytoplasm, the pre-miRNA is further processed by Dicer,another RNaseIII enzyme, to produce a duplex of ˜22 bp that isstructurally identical to an siRNA duplex (Hutvagner et al., Science293: 834-838, 2001). The binding of protein components of theRNA-induced silencing complex (RISC), or RISC cofactors, to the duplexresults in incorporation of the mature, single-stranded miRNA into aRISC or RISC-like protein complex, whereas the other strand of theduplex is degraded (Bartel, Cell 116: 281-297, 2004).

The miR-30 architecture can be used to express miRNAs or siRNAs from polII promoter-based expression plasmids. See also Zeng et al., Methods inEnzymology 392: 371-380, 2005 (incorporated herein by reference).

FIG. 2B of Zeng (supra) shows the predicted secondary structure of themiR-30 precursor hairpin (“the miR-30 cassette”). Boxed are extranucleotides that were added originally for subcloning purposes (Zeng andCullen, RNA 9: 112-123, 2003; Zeng et al., Mol. Cell. 9: 1327-1333,2002). They represent XhoI-BgIII sites at the 50 end and BamHI-XhoIsites at the 30 end. These appended nucleotides extend the minimalmiR-30 precursor stem shown by several basepairs, similar to the in vivosituation where the primary miR-30 precursor is transcribed from itsgenomic locus (Lee et al., Nature 425: 415-419, 2003), and an extendedstem of at least 5 bp is essential for efficient miR-30 production.Based on the numbering in FIG. 2B, mature miR-30 is encoded bynucleotides 44 to 65 and anti-miR-30 by nucleotides 3 to 25 of thisprecursor. In the simplest expression setting, the cytomegalovirus (CMV)immediate early enhancer/promoter may be used to transcribe the miR-30cassette. The cassette is preceded by a leader sequence of approximately100 nt and followed by approximately 170 nt before the polyadenylationsite (Zeng et al., Mol. Cell. 9: 1327-1333, 2002). These lengths arearbitrary and can be longer or shorter. Mature 22-nt miR-30 can be madefrom such constructs.

Several other authentic miRNAs have been over-expressed by usinganalogous RNA pol II-based expression vectors or even pol III-dependentpromoters (Chen et al., Science 303: 83-86, 2004; Zeng and Cullen, RNA9: 112-123, 2003). Expression simply requires the insertion of theentire predicted miRNA precursor stem-loop structure into the expressionvector at an arbitrary location. Because the actual extent of theprecursor stem loop can sometimes be difficult to accurately predict, itis generally appropriate to include ˜50 bp of flanking sequence on eachside of the predicted ˜80-nt miRNA stem-loop precursor to be sure thatall cis-acting sequences necessary for accurate and efficient Droshaprocessing are included (Chen et al., Science 303: 83-86, 2004).

In an exemplary embodiment, to make the miR-30 expression cassette, thesequence from +1 to 65 (excluding the 15-nt terminal loop of the miR-30cassette, FIG. 2B of Zeng) may be replaced as follows: the sequence fromnucleotides 39 to 61, which is perfectly complementary to a target genesequence, will act as the active strand during RNAi. The sequence fromnucleotides 2 to 23 is thus designed to preserve the double-strandedstem in the miR-30-target cassette, but nucleotide +1 is now a C, tocreate a mismatch with nucleotide 61, a U, just like nucleotides 1 and65 in the miR-30 cassette (FIG. 2B). Because the 30 arm of the stem(miR-30-target) is the active component for RNAi, changes in the 50 armof the stem will not affect RNAi specificity. A 2-nt bulge may bepresent in the stem region of the authentic miR-30 precursor (FIG. 2B ofZeng). A break in the helical nature of the RNA stem may help ward offnonspecific effects, such as induction of an interferon response (Bridgeet al., Nat. Genet. 34: 263-264, 2003) in expressing cells. This may bewhy miRNA precursors almost invariably contain bulges in the predictedstem. The miR-30 cassette in FIG. 2A of Zeng is then substituted withthe miR-30-target cassette, and the resulting expression plasmid can betransfected into target cells.

The use of pol II promoters, especially when coupled with an inducibleexpression system (such as the TetOFF system of Clontech) offersflexibility in regulating the production of miRNAs in cultured cells orin vivo. Selection of stable cell lines leads to less leaky expressionin the absence of the activator or presence of doxycycline, andtherefore a stronger induction.

In certain embodiments, it would be advantageous if the antisensestrand, for example, of the above miR-30-target construct ispreferentially made as a mature miRNA, because its opposite strand doesnot have any known target. The relative basepairing stability at the 50ends of an siRNA duplex is a strong determinant of which strand will beincorporated into RISC and hence be active in RNAi; the strand whose 50end has a weaker hydrogen bonding pattern is preferentially incorporatedinto RISC, the RNAi effector complex (Khvorova et al., Cell 115:209-216, 2003; Schwarz et al., Cell 115: 208-299, 2003). This sameprinciple can also be applied to the design of DNA vector-based siRNAexpression strategies, including the one described here. However, forartificial miRNAs, the fact that the internal cleavage sites by Droshaand Dicer cannot be precisely predicted at present adds a degree ofuncertainty as a 1- or 2-nt shift in the cleavage site can generaterather different hydrogen bonding patterns at the 50 ends of theresulting duplex, thus changing which strand of the duplex intermediateis incorporated into RISC. This is in contrast to the situation withsynthetic siRNA duplexes, which have defined ends. On the other hand,any minor heterogeneity at the ends of an artificial miRNA duplexintermediate might not be a problem, as the miRNAs would still beperfectly complementary to their target.

The role of internal loop, stem length, and the surrounding sequences onthe expression of miRNAs from miR-30-derived cassettes may also besystematically examined to optimize expression of the miR-based shRNA.Such analyses may suggest design elements that would maximize the yieldof the intended RNA products. On the other hand, some heterogeneitycould be inevitable. In addition to the 50-end rule, specific residuesat some positions within an siRNA may also enhance siRNA function(Reynolds et al., Nat. Biotech. 22: 326-330, 2004).

In general, picking a target region with more than 50% AU content anddesigning a weak 50 end base pair on the antisense strand would be agood starting point in the design of any artificial miRNA/siRNAexpression plasmid (Khvorova et al., Cell 115: 209-216, 2003; Reynoldset al., Nat. Biotech. 22: 326-330, 2004; Schwarz et al., Cell 115:208-299, 2003).

In certain embodiments, expression of the miR-30 cassette may be in theantisense orientation, especially when the cassette is to be used inlentiviral or retroviral vectors. This is partly because miRNAprocessing may result in the degradation of the remainder of the primarymiRNA transcript.

In other embodiments, vectors may contain inserts expressing more thanone miRNAs. In such constructs, the fact that each miRNA stem-loopprecursor is independently excised from the primary transcript by Droshacleavage to give rise to a pre-miRNA allows simultaneous expression ofseveral artificial or authentic miRNAs by a tandem array on a precursorRNA transcript.

Genome wide libraries of shRNAs based on the miR30 precursor RNA havealso been generated. Each member of such libraries target specific humanor mouse genes, and may be readily converted to the vectors/expressionsystems of the instant invention. The following section describes thedesign of such libraries.

Paddison et al. (Nature Methods 1(2): 163-67, 2004, incorporated hereinby reference) have described a genome-wise library of shRNAs based onthe miR30 precursor RNA, which may be adapted for use in the instantinvention. The described vector pSHAG-MAGIC2 (pSM2) is roughlyequivalent to pSHAG-MAGIC1 as described in Paddison et al. Methods Mol.Biol. 265: 85-100 (2004), incorporated herein by reference. The fewnotable exceptions include: the new cloning strategy is based on the useof a single oligonucleotide that contains the hairpin and common 5′ and3′ ends as a PCR template (see FIG. 2 of Paddison, Nature Methods 1(2):163-67, 2004). The resulting PCR product is then cloned into the hairpincloning site of the pSM2 vector, which drives miR-30-styled hairpins bythe human U6 promoter. Inserts from this library may be excised (seeExample below) and cloned into the instant vectors for Pol II-drivenexpression of the same miR-30-styled hairpins. This allows the instantmethods to be coupled with the existing library of miR-30-styleconstructs that contains most human and mouse genes.

Paddison also describes the detailed methods for designing 22-nucleotidesequences (targeting a target gene) that can be inserted into theprecursor miRNA, PCR protocols for amplification, and relevant criticalsteps and trouble-shootings, etc. (all incorporated herein byreference).

MicroRNAs (including the siRNA products and artificial microRNAs as wellas endogenous microRNAs) have potential for use as therapeutics as wellas research tools, e.g. analyzing gene function. As a general method,the mature microRNA (miR) of the invention, especially those non-miR-30based microRNA constructs of the invention may also be producedaccording to the following description.

In certain embodiments, the methods for efficient expression of microRNAinvolve the use of a precursor microRNA molecule having a microRNAsequence in the context of microRNA flanking sequences. The precursormicroRNA is composed of any type of nucleic acid based molecule capableof accommodating the microRNA flanking sequences and the microRNAsequence. Examples of precursor microRNAs and the individual componentsof the precursor (flanking sequences and microRNA sequence) are providedherein. The invention, however, is not limited to the examples provided.The invention is based, at least in part, on the discovery of animportant component of precursor microRNAs, that is, the microRNAflanking sequences. The nucleotide sequence of the precursor and itscomponents may vary widely.

In one aspect a precursor microRNA molecule is an isolated nucleic acidincluding microRNA flanking sequences and having a stem-loop structurewith a microRNA sequence incorporated therein. An “isolated molecule” isa molecule that is free of other substances with which it is ordinarilyfound in nature or in vivo systems to an extent practical andappropriate for its intended use. In particular, the molecular speciesare sufficiently free from other biological constituents of host cellsor if they are expressed in host cells they are free of the form orcontext in which they are ordinarily found in nature. For instance, anucleic acid encoding a precursor microRNA having homologous microRNAsequences and flanking sequences may ordinarily be found in a host cellin the context of the host cell genomic DNA. An isolated nucleic acidencoding a microRNA precursor may be delivered to a host cell, but isnot found in the same context of the host genomic DNA as the naturalsystem. Alternatively, an isolated nucleic acid is removed from the hostcell or present in a host cell that does not ordinarily have such anucleic acid sequence. Because an isolated molecular species of theinvention may be admixed with a pharmaceutically-acceptable carrier in apharmaceutical preparation or delivered to a host cell, the molecularspecies may comprise only a small percentage by weight of thepreparation or cell. The molecular species is nonetheless isolated inthat it has been substantially separated from the substances with whichit may be associated in living systems.

An “isolated precursor microRNA molecule” is one which is produced froma vector having a nucleic acid encoding the precursor microRNA. Thus,the precursor microRNA produced from the vector may be in a host cell orremoved from a host cell. The isolated precursor microRNA may be foundwithin a host cell that is capable of expressing the same precursor. Itis nonetheless isolated in that it is produced from a vector and, thus,is present in the cell in a greater amount than would ordinarily beexpressed in such a cell.

The term “nucleic acid” is used to mean multiple nucleotides (i.e.molecules comprising a sugar (e.g. ribose or deoxyribose) linked to aphosphate group and to an exchangeable organic base, which is either asubstituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U))or a substituted purine (e.g. adenine (A) or guanine (G)). The termshall also include polynucleosides (i.e. a polynucleotide minus thephosphate) and any other organic base containing polymer. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymidine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and othernaturally and non-naturally occurring nucleobases, substituted andunsubstituted aromatic moieties. Other such modifications are well knownto those of skill in the art. Thus, the term nucleic acid alsoencompasses nucleic acids with substitutions or modifications, such asin the bases and/or sugars.

“MicroRNA flanking sequence” as used herein refers to nucleotidesequences including microRNA processing elements. MicroRNA processingelements are the minimal nucleic acid sequences which contribute to theproduction of mature microRNA from precursor microRNA. Often theseelements are located within a 40 nucleotide sequence that flanks amicroRNA stem-loop structure. In some instances the microRNA processingelements are found within a stretch of nucleotide sequences of between 5and 4,000 nucleotides in length that flank a microRNA stem-loopstructure.

Thus, in some embodiments the flanking sequences are 5-4,000 nucleotidesin length. As a result, the length of the precursor molecule may be, insome instances at least about 150 nucleotides or 270 nucleotides inlength. The total length of the precursor molecule, however, may begreater or less than these values. In other embodiments the minimallength of the microRNA flanking sequence is 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200 and any integer there between. In otherembodiments the maximal length of the microRNA flanking sequence is2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900,3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,9004,000 and any integer there between.

The microRNA flanking sequences may be native microRNA flankingsequences or artificial microRNA flanking sequences. A native microRNAflanking sequence is a nucleotide sequence that is ordinarily associatedin naturally existing systems with microRNA sequences, i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo. Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking to microRNAsequences in naturally existing systems. The artificial microRNAflanking sequences may be flanking sequences found naturally in thecontext of other microRNA sequences. Alternatively they may be composedof minimal microRNA processing elements which are found within naturallyoccurring flanking sequences and inserted into other random nucleic acidsequences that do not naturally occur as flanking sequences or onlypartially occur as natural flanking sequences.

The microRNA flanking sequences within the precursor microRNA moleculemay flank one or both sides of the stem-loop structure encompassing themicroRNA sequence. Thus, one end (i.e., 5′) of the stem-loop structuremay be adjacent to a single flanking sequence and the other end (i.e.,3′) of the stem-loop structure may not be adjacent to a flankingsequence. Preferred structures have flanking sequences on both ends ofthe stem-loop structure. The flanking sequences may be directly adjacentto one or both ends of the stem-loop structure or may be connected tothe stem-loop structure through a linker, additional nucleotides orother molecules.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (stem portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion). The terms “hairpin” and “fold-back” structures are also usedherein to refer to stem-loop structures. Such structures are well knownin the art and the term is used consistently with its known meaning inthe art. The actual primary sequence of nucleotides within the stem-loopstructure is not critical to the practice of the invention as long asthe secondary structure is present. As is known in the art, thesecondary structure does not require exact base-pairing. Thus, the stemmay include one or more base mismatches. Alternatively, the base-pairingmay be exact, i.e. not include any mismatches.

In some instances the precursor microRNA molecule may include more thanone stem-loop structure. The multiple stem-loop structures may be linkedto one another through a linker, such as, for example, a nucleic acidlinker or by a microRNA flanking sequence or other molecule or somecombination thereof.

In an alternative embodiment, useful interfering RNAs can be designedwith a number of software programs, e.g., the OligoEngine siRNA designtool available at wwv.olioengine.com. The siRNAs of this invention mayrange about, e.g., 19-29 basepairs in length for the double-strandedportion. In some embodiments, the siRNAs are hairpin RNAs having anabout 19-29 bp stem and an about 4-34 nucleotide loop. Preferred siRNAsare highly specific for a region of the target gene and may comprise anyabout 19-29 bp fragment of a target gene mRNA that has at least one,preferably at least two or three, bp mismatch with a nontargetgene-related sequence. In some embodiments, the preferred siRNAs do notbind to RNAs having more than 3 mismatches with the target region.

III. Expression Vectors and Host Cells

The invention also includes vectors for producing precursor microRNAmolecules. Generally these vectors include a sequence encoding aprecursor microRNA and (in vivo) expression elements. The expressionelements include at least one promoter, such as a Pol II promoter, whichmay direct the expression of the operably linked microRNA precursor(e.g. the shRNA encoding sequence). The vector or primary transcript isfirst processed to produce the stem-loop precursor molecule. Thestem-loop precursor is then processed to produce the mature microRNA.

RNA polymerase III (Pol III) transcription units normally encode thesmall nuclear RNA U6 (see Tran et al., BMC Biotechnology 3: 21, 2003,incorporate herein by reference), or the human RNAse P RNA Hi. However,RNA polymerase II (Pol II) transcription units (e.g., units containing aCMV promoter) is preferred for use with inducible expression. It will beappreciated that in the vectors of the invention, the subject shRNAencoding sequence may be operably linked to a variety of otherpromoters.

In some embodiments, the promoter is a type II tRNA promoter such as thetRNAVa promoter and the tRNAmet promoter. These promoters may also bemodified to increase promoter activity. In addition, enhancers can beplaced near the promoter to enhance promoter activity. Pot II enhancermay also be used for Pol III promoters. For example, an enhancer fromthe CMV promoter can be placed near the U6 promoter to enhance U6promoter activity (Xia et al., Nuc Acids Res 31, 2003).

In certain embodiments, the subject Pol II promoters are induciblepromoters. Exemplary inducible Pol II systems are available fromInvitrogen, e.g., the GeneSwitch™ or T-REx™ systems; from Clontech (PaloAlto, Calif.), e.g., the TetON and TetOFF systems.

An exemplary Tet-responsive promoter is described in WO 04/056964A2(incorporated herein by reference). See, for example, FIG. 1 of WO04/056964A2. In one construct, a Tet operator sequence (TetOp) isinserted into the promoter region of the vector. TetOp is preferablyinserted between the PSE and the transcription initiation site, upstreamor downstream from the TATA box. In some embodiments, the TetOp isimmediately adjacent to the TATA box. The expression of the subjectshRNA encoding sequence is thus under the control of tetracycline (orits derivative doxycycline, or any other tetracycline analogue).Addition of tetracycline or Dox relieves repression of the promoter by atetracycline repressor that the host cells are also engineered toexpress.

In the TetOFF system, a different tet transactivator protein isexpressed in the tetOFF host cell. The difference is that Tet/Dox, whenbind to an activator protein, is now required for transcriptionalactivation. Thus such host cells expressing the activator will onlyactivate the transcription of an shRNA encoding sequence from a TetOFFpromoter at the presence of Tet or Dox.

An alternative inducible promoter is a lac operator system, asillustrated in FIG. 2A of WO 04/056964 A2 (incorporated by reference).Briefly, a Lac operator sequence (LacO) is inserted into the promoterregion. The LacO is preferably inserted between the PSE and thetranscription initiation site, upstream or downstream of the TATA box.In some embodiments, the LacO is immediately adjacent to the TATA box.The expression of the RNAi molecule (shRNA encoding sequence) is thusunder the control of IPTG (or any analogue thereof). Addition of IPTGrelieves repression of the promoter by a Lac repressor (i.e., the LacIprotein) that the host cells are also engineered to express. Since theLac repressor is derived from bacteria, its coding sequence may beoptionally modified to adapt to the codon usage by mammaliantranscriptional systems and to prevent methylation. In some embodiments,the host cells comprise (i) a first expression construct containing agene encoding a Lac repressor operably linked to a first promoter, suchas any tissue or cell type specific promoter or any general promoter,and (ii) a second expression construct containing the dsRNA-codingsequence operably linked to a second promoter that is regulated by theLac repressor and IPTG. Administration of IPTG results in expression ofdsRNA in a manner dictated by the tissue specificity of the firstpromoter.

Yet another inducible system, a LoxP-stop-LoxP system, is illustrated inFIGS. 3A-3E of WO 04/056964 A2 (incorporated by reference). The RNAivector of this system contains a LoxP-Stop-LoxP cassette before thehairpin or within the loop of the hairpin. Any suitable stop sequencefor the promoter can be used in the cassette. One version of the LoxPStop-LoxP system for Pol II is described in, e.g., Wagner et al.,Nucleic Acids Research 25:4323-4330, 1997. The “Stop” sequences (such asthe one described in Wagner, sierra, or a run of five or more Tnucleotides) in the cassette prevent the RNA polymerase III fromextending an RNA transcript beyond the cassette. Upon introduction of aCre recombinase, however, the LoxP sites in the cassette recombine,removing the Stop sequences and leaving a single LoxP site. Removal ofthe Stop sequences allows transcription to proceed through the hairpinsequence, producing a transcript that can be efficiently processed intoan open-ended, interfering dsRNA. Thus, expression of the RNAi moleculeis induced by addition of Cre.

In some embodiments, the host cells contain a Cre-encoding transgeneunder the control of a constitutive, tissue-specific promoter. As aresult, the interfering RNA can only be inducibly expressed in atissue-specific manner dictated by that promoter. Tissue-specificpromoters that can be used include, without limitation: a tyrosinasepromoter or a TRP2 promoter in the case of melanoma cells andmelanocytes; an MMTV or WAP promoter in the case of breast cells and/orcancers; a Villin or FABP promoter in the case of intestinal cellsand/or cancers; a RIP promoter in the case of pancreatic beta cells; aKeratin promoter in the case of keratinocytes; a Probasin promoter inthe case of prostatic epithelium; a Nestin or GFAP promoter in the caseof CNS cells and/or cancers; a Tyrosine Hydroxylase, S100 promoter orneurofilament promoter in the case of neurons; the pancreas-specificpromoter described in Edlund et al., Science 230: 912-916, 1985; a Claracell secretory protein promoter in the case of lung cancer; and an Alphamyosin promoter in the case of cardiac cells.

Cre expression also can be controlled in a temporal manner, e.g., byusing an inducible promoter, or a promoter that is temporally restrictedduring development such as Pax3 or Protein O (neural crest), Hoxal(floorplate and notochord), Hoxb6 (extraembryonic mesoderm, lateralplate and limb mesoderm and midbrain-hindbrain junction), Nestin(neuronal lineage), GFAP (astrocyte lineage), Lck (immature thymocytes).Temporal control also can be achieved by using an inducible form of Cre.For example, one can use a small molecule controllable Cre fusion, forexample a fusion of the Cre protein and the estrogen receptor (ER) orwith the progesterone receptor (PR). Tamoxifen or RU486 allow the Cre-ERor Cre-PR fusion, respectively, to enter the nucleus and recombine theLoxP sites, removing the LoxP Stop cassette. Mutated versions of eitherreceptor may also be used. For example, a mutant Cre-PR fusion proteinmay bind RU486 but not progesterone. Other exemplary Cre fusions are afusion of the Cre protein and the glucocorticoid receptor (GR). NaturalGR ligands include corticosterone, cortisol, and aldosterone. Mutantversions of the GR receptor, which respond to, e.g., dexamethasone,triamcinolone acetonide, and/or RU38486, may also be fused to the Creprotein.

In certain embodiments, additional transcription units may be present 3′to the shRNA portion. For example, an internal ribosomal entry site(IRES) may be positioned downstream of the shRNA insert, thetranscription of which is under the control of a second promoter, suchas the PGK promoter. The IRES sequence may be used to direct theexpression of a operably linked second gene, such as a reporter gene(e.g., a fluorescent protein such as GFP, BFP, YFP, etc., an enzyme suchas luciferase (Promega), etc.). The reporter gene may serve as anindication of infection/transfection, and the efficiency and/or amountof mRNA transcription of the shRNA-IRES-reporter cassette/insert.Optionally, one or more selectable markers (such as puromycin resistancegene, neomycin resistance gene, hygromycin resistance gene, zeocinresistance gene, etc.) may also be present on the same vector, and areunder the transcriptional control of the second promoter. Such markersmay be useful for selecting stable integration of the vector into a hostcell genome.

Certain exemplary vectors useful for expressing the precursor microRNAsare shown in the examples. Thus the invention encompasses the nucleotidesequence of such vectors as well as variants thereof.

In general, variants typically will share at least 40% nucleotideidentity with any of the described vectors, in some instances, willshare at least 50% nucleotide identity; and in still other instances,will share at least 60% nucleotide identity. The preferred variants haveat least 70% sequence homology. More preferably the preferred variantshave at least 80% and, most preferably, at least 90% sequence homologyto the described sequences.

Variants with high percentage sequence homology can be identified, forexample, using stringent hybridization conditions. The term “stringentconditions”, as used herein, refers to parameters with which the art isfamiliar. More specifically, stringent conditions, as used herein, referto hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15Msodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetraacetic acid. After hybridization, the membrane towhich the DNA is transferred is washed at 2×SSC at room temperature andthen at 0.1×SSC/0.1×SDS at 65° C. There are other conditions, reagents,and so forth which can be used, which result in a similar degree ofstringency. Such variants may be further subject to functional testingsuch that variants that substantially preserve the desired/relevantfunction of the original vectors are selected/identified.

The “in vivo expression elements” are any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient expression of the nucleic acid toproduce the precursor microRNA. The in vivo expression element may, forexample, be a mammalian or viral promoter, such as a constitutive orinducible promoter or a tissue specific promoter. Constitutive mammalianpromoters include, but are not limited to, polymerase II promoters aswell as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase,and β-actin. Exemplary viral promoters which function constitutively ineukaryotic cells include, for example, promoters from the simian virus,papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofmoloney leukemia virus and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as in vivoexpression element of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription in the presence of certain metal ions. Other induciblepromoters are known to those of ordinary skill in the art.

One useful inducible expression system that can be adapted for use inthe instant invention is the Tet-responsive system, including both theTetON and TetOFF embodiments.

TetOn system is a commercially available inducible expression systemfrom Clontech Inc. This is of particular interest because current siRNAexpression systems utilize pol III promoters, which are difficult toadapt for inducible expression. The Clontech TetON system includes thepRev-TRE vector, which can be packaged into retrovirus and used toinfect a Tet-On cell line expressing the reverse tetracycline-controlledtransactivator (rtTA). Once introduced into the TetON host cell, theshRNA insert can then be inducibly expressed in response to varyingconcentrations of the tetracycline derivate doxycycline (Dox).

In general, the in vivo expression element shall include, as necessary,5′ non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription. They optionally include enhancer sequencesor upstream activator sequences as desired.

Vectors include, but are not limited to, plasmids, phagemids, viruses,other vehicles derived from viral or bacterial sources that have beenmanipulated by the insertion or incorporation of the nucleic acidsequences for producing the precursor microRNA, and free nucleic acidfragments which can be attached to these nucleic acid sequences. Viraland retroviral vectors are a preferred type of vector and include, butare not limited to, nucleic acid sequences from the following viruses:retroviruses, such as: Moloney murine leukemia virus; Murine stem cellvirus, Harvey murine sarcoma virus; murine mammary tumor virus; Roussarcoma virus; adenovirus; adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpesviruses; vaccinia viruses; polio viruses; lentiviruses; and RNA virusessuch as any retrovirus. One can readily employ other unnamed vectorsknown in the art.

Viral vectors are generally based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the nucleic acidsequence of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Retroviruses have been approved for human gene therapy trials.Genetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of nucleic acids in vivo. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell lined with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W.H. Freeman Co., New York (1990) andMurry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

Exemplary vectors are disclosed herein and in US 2005/0075492 A2(incorporated herein by reference) and WO 04/056964 A2 (incorporatedherein by reference).

The invention also encompasses host cells transfected with the subjectvectors, especially host cell lines with stably integrated shRNAconstructs. In certain embodiments, the subject host cell contains onlya single copy of the integrated construct expressing the desired shRNA(optionally under the control of an inducible and/or tissue specificpromoter). Host cells include for instance, cells (such as primarycells) and cell lines, e.g. prokaryotic (e.g., E. coli), and eukaryotic(e.g., dendritic cells, CHO cells, COS cells, yeast expression systemsand recombinant baculovirus expression in insect cells, etc.). Exemplarycells include: NIH3T3 cells, MEFs, 293 or 293T cells, CHO cells,hematopoietic stern/progenitor cells, cancer cells, etc.

IV. Methods of Using

In certain aspects, methods of the invention comprise contacting andintroducing into a target cell with a subject vector capable ofexpressing a precursor microRNA as described herein, to regulate theexpression of a target gene in the cell. The vector produces themicroRNA transcript, which is then processed into precursor microRNA inthe cell, which is then processed to produce the mature functionalmicroRNA which is capable of altering accumulation of a target proteinin the target cell. Accumulation of the protein may be effected in anumber of different ways. For instance the microRNA may directly orindirectly affect translation or may result in cleavage of the mRNAtranscript or even effect stability of the protein being translated fromthe target mRNA. MicroRNA may function through a number of differentmechanisms. The methods and products of the invention are not limited toany one mechanism. The method may be performed in vitro, e.g., forstudying gene function, ex vivo or in vivo, e.g. for therapeuticpurposes.

An “ex vivo” method as used herein is a method which involves isolationof a cell from a subject, manipulation of the cell outside of the body,and reimplantation of the manipulated cell into the subject. The ex vivoprocedure may be used on autologous or heterologous cells, but ispreferably used on autologous cells. In preferred embodiments, the exvivo method is performed on cells that are isolated from bodily fluidssuch as peripheral blood or bone marrow, but may be isolated from anysource of cells. When returned to the subject, the manipulated cell willbe programmed for cell death or division, depending on the treatment towhich it was exposed. Ex vivo manipulation of cells has been describedin several references in the art, including Engleman, E. G., 1997,Cytotechnology, 25:1; Van Schooten, W., et al., 1997, Molecular MedicineToday, June, 255; Steinman, R. M., 1996, Experimental Hematology, 24,849; and Gluckman, J. C., 1997, Cytokines, Cellular and MolecularTherapy, 3:187. The ex vivo activation of cells of the invention may beperformed by routine ex vivo manipulation steps known in the art. Invivo methods are also well known in the art. The invention thus isuseful for therapeutic purposes and also is useful for research purposessuch as testing in animal or in vitro models of medical, physiologicalor metabolic pathways or conditions.

The ex vivo and in vivo methods are performed on a subject. A “subject”shall mean a human or non-human mammal, including but not limited to, adog, cat, horse, cow, pig, sheep, goat, primate, rat, and mouse, etc.

In some instances the mature microRNA is expressed at a level sufficientto cause at least a 2-fold, or in some instances, a 10 fold reduction inaccumulation of the target protein. The level of accumulation of atarget protein may be assessed using routine methods known to those ofskill in the art. For instance, protein may be isolated from a targetcell and quantitated using Western blot analysis or other comparablemethodologies, optionally in comparison to a control. Protein levels mayalso be assessed using reporter systems or fluorescently labeledantibodies. In other embodiments, the mature microRNA is expressed at alevel sufficient to cause at least a 2, 5, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, or 100 fold reduction in accumulation of thetarget protein. The “fold reduction” may be assessed using any parameterfor assessing a quantitative value of protein expression. For instance,a quantitative value can be determined using a label i.e. fluorescent,radioactive linked to an antibody. The value is a relative value that iscompared to a control or a known value.

Different microRNA sequences have different levels of expression ofmature microRNA and thus have different effects on target mRNA and/orprotein expression. For instance, in some cases a microRNA may beexpressed at a high level and may be very efficient such that theaccumulation of the target protein is completely or near completelyblocked. In other instances the accumulation of the target protein maybe only reduced slightly over the level that would ordinarily beexpressed in that cell at that time under those conditions in theabsence of the mature microRNA. Complete inhibition of the accumulationof the target protein is not essential, for example, for therapeuticpurposes. In many cases partial or low inhibition of accumulation mayproduce a preferred phenotype. The actual amount that is useful willdepend on the particular cell. type, the stage of differentiation,conditions to which the cell is exposed, the modulation of other targetproteins, etc.

The microRNAs may be used to knock down gene expression in vertebratecells for gene-function studies, including target-validation studiesduring the development of new pharmaceuticals, as well as thedevelopment of human disease models and therapies, and ultimately, humangene therapies.

The methods of the invention are useful for treating any type of“disease”, “disorder” or “condition” in which it is desirable to reducethe expression or accumulation of a particular target protein(s).Diseases include, for instance, but are not limited to, cancer,infectious disease, cystic fibrosis, blood disorders, including leukemiaand lymphoma, spinal muscular dystrophy, early-onset Parkinsonism(Waisman syndrome) and X-linked mental retardation (MRx3).

Cancers include but are not limited to biliary tract cancer; bladdercancer; breast cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancerincluding colorectal carcinomas; endometrial cancer; esophageal cancer;gastric cancer; head and neck cancer; hematological neoplasms includingacute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer including small cell lung cancer and non-smallcell lung cancer; lymphomas including Hodgkin's disease and lymphocyticlymphomas; neuroblastomas; oral cancer including squamous cellcarcinoma; osteosarcomas; ovarian cancer including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreatic cancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, synovialsarcoma and osteosarcoma; skin cancer including melanomas, Kaposi'ssarcoma, basocellular cancer, and squamous cell cancer; testicularcancer including germinal tumors such as seminoma, non-seminoma(teratomas, choriocarcinomas), stromal tumors, and germ cell tumors;thyroid cancer including thyroid adenocarcinoma and medullar carcinoma;transitional cancer and renal cancer including adenocarcinoma and Wilmstumor.

An infectious disease, as used herein, is a disease arising from thepresence of a foreign microorganism in the body. A microbial antigen, asused herein, is an antigen of a microorganism. Microorganisms includebut are not limited to, infectious virus, infectious bacteria, andinfectious fungi.

Examples of infectious virus include but are not limited to:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-I (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema palladium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax andToxoplasma gondii.

The vectors of this invention can be delivered into host cells via avariety of methods, including but not limited to, liposome fusion(transposomes), infection by viral vectors, and routine nucleic acidtransfection methods such as electroporation, calcium phosphateprecipitation and microinjection. In some embodiments, the vectors areintegrated into the genome of a transgenic animal (e.g., a mouse, arabbit, a hamster, or a nonhuman primate). Diseased or disease-pronecells containing these vectors can be used as a model system to studythe development, maintenance, or progression of a disease that isaffected by the presence or absence of the interfering RNA.

Expression of the miRNA/siRNA introduced into a target cell may beconfirmed by art-recognized techniques, such as Northern blotting usinga nucleic acid probe. For cell lines that are more difficult totransfect, more extracted RNA can be used for analyses, optionallycoupled with exposing the film longer. Once expression of themiRNA/siRNA is confirmed, the DNA construct can then be tested for RNAiefficacy against a cotransfected construct encoding the target proteinor directly against an endogenous target. In the latter case, onepreferably should have a clear idea of transfection efficiency and ofthe half-life of the target protein before performing the experiment.

V. Pharmaceutical Use and Methods of Administration

In one aspect, the invention provides a method of administering any ofthe compositions described herein to a subject. When administered, thecompositions are applied in a therapeutically effective,pharmaceutically acceptable amount as a pharmaceutically acceptableformulation. As used herein, the term “pharmaceutically acceptable” isgiven its ordinary meaning. Pharmaceutically acceptable compounds aregenerally compatible with other materials of the formulation and are notgenerally deleterious to the subject. Any of the compositions of thepresent invention may be administered to the subject in atherapeutically effective dose. A “therapeutically effective” or an“effective” as used herein means that amount necessary to delay theonset of, inhibit the progression of, halt altogether the onset orprogression of, diagnose a particular condition being treated, orotherwise achieve a medically desirable result, i.e., that amount whichis capable of at least partially preventing, reversing, reducing,decreasing, ameliorating, or otherwise suppressing the particularcondition being treated. A therapeutically effective amount can bedetermined on an individual basis and will be based, at least in part,on consideration of the species of mammal, the mammal's age, sex, size,and health; the compound and/or composition used, the type of deliverysystem used; the time of administration relative to the severity of thedisease; and whether a single, multiple, or controlled-release doseregiment is employed. A therapeutically effective amount can bedetermined by one of ordinary skill in the art employing such factorsand using no more than routine experimentation.

The terms “treat,” “treated,” “treating,” and the like, when usedherein, refer to administration of the systems and methods of theinvention to a subject, which may, for example, increase the resistanceof the subject to development or further development of cancers, toadministration of the composition in order to eliminate or at leastcontrol a cancer or a infectious disease, and/or to reduce the severityof the cancer or infectious disease, or symptoms thereof. Such termsalso include prevention of disease/condition in, for example,subjects/individuals predisposed to such diseases/conditions, or at highrisk of developing such diseases/conditions.

When administered to a subject, effective amounts will depend on theparticular condition being treated and the desired outcome. Atherapeutically effective dose may be determined by those of ordinaryskill in the art, for instance, employing factors such as those furtherdescribed below and using no more than routine experimentation.

In administering the systems and methods of the invention to a subject,dosing amounts, dosing schedules, routes of administration, and the likemay be selected so as to affect known activities of these systems andmethods. Dosage may be adjusted appropriately to achieve desired druglevels, local or systemic, depending upon the mode of administration.The doses may be given in one or several administrations per day. As oneexample, if daily doses are required, daily doses may be from about 0.01mg/kg/day to about 1000 mg/kg/day, and in some embodiments, from about0.1 to about 100 mg/kg/day or from about 1 mg/kg/day to about 10mg/kg/day. Parental administration, in some cases, may be from one toseveral orders of magnitude lower dose per day, as compared to oraldoses. For example, the dosage of an active compound when parentallyadministered may be between about 0.1 micrograms/kg/day to about 10mg/kg/day, and in some embodiments, from about 1 microgram/kg/day toabout 1 mg/kg/day or from about 0.01 mg/kg/day to about 0.1 mg/kg/day.

In some embodiments, the concentration of the active compound(s), ifadministered systemically, is at a dose of about 1.0 mg to about 2000 mgfor an adult of 70 kg body weight, per day. In other embodiments, thedose is about 10 mg to about 1000 mg/70 kg/day. In yet otherembodiments, the dose is about 100 mg to about 500 mg/70 kg/day.Preferably, the concentration, if applied topically, is about 0.1 mg toabout 500 mg/gm of ointment or other base, more preferably about 1.0 mgto about 100 mg/gm of base, and most preferably, about 30 mg to about 70mg/gm of base. The specific concentration partially depends upon theparticular composition used, as some are more effective than others. Thedosage concentration of the composition actually administered isdependent at least in part upon the particular physiological responsebeing treated, the final concentration of composition that is desired atthe site of action, the method of administration, the efficacy of theparticular composition, the longevity of the particular composition, andthe timing of administration relative to the severity of the disease.Preferably, the dosage form is such that it does not substantiallydeleteriously affect the mammal. The dosage can be determined by one ofordinary skill in the art employing such factors and using no more thanroutine experimentation.

In the event that the response of a particular subject is insufficientat such doses, even higher doses (or effectively higher doses by adifferent, more localized delivery route) may be employed to the extentthat subject tolerance permits. Multiple doses per day are alsocontemplated in some cases to achieve appropriate systemic levels withinthe subject or within the active site of the subject. In some cases,dosing amounts, dosing schedules, routes of administration, and the likemay be selected as described herein, whereby therapeutically effectivelevels for the treatment of cancer are provided.

In certain embodiments where cancers are being treated, a composition ofthe invention may be administered to a subject who has a family historyof cancer, or to a subject who has a genetic predisposition for cancer.In other embodiments, the composition is administered to a subject whohas reached a particular age, or to a subject more likely to get cancer.In yet other embodiments, the compositions is administered to subjectswho exhibit symptoms of cancer (e.g., early or advanced). In still otherembodiments, the composition may be administered to a subject as apreventive measure. In some embodiments, the inventive composition maybe administered to a subject based on demographics or epidemiologicalstudies, or to a subject in a particular field or career.

Administration of a composition of the invention to a subject may beaccomplished by any medically acceptable method which allows thecomposition to reach its target. The particular mode selected willdepend of course, upon factors such as those previously described, forexample, the particular composition, the severity of the state of thesubject being treated, the dosage required for therapeutic efficacy,etc. As used herein, a “medically acceptable” mode of treatment is amode able to produce effective levels of the active compound(s) of thecomposition within the subject without causing clinically unacceptableadverse effects.

Any medically acceptable method may be used to administer a compositionto the subject. The administration may be localized (i.e., to aparticular region, physiological system, tissue, organ, or cell type) orsystemic, depending on the condition being treated. For example, thecomposition may be administered orally, vaginally, rectally, buccally,pulmonary, topically, nasally, transdermally, through parenteralinjection or implantation, via surgical administration, or any othermethod of administration where suitable access to a target is achieved.Examples of parenteral modalities that can be used with the inventioninclude intravenous, intradermal, subcutaneous, intracavity,intramuscular, intraperitoneal, epidural, or intrathecal. Examples ofimplantation modalities include any implantable or injectable drugdelivery system. Oral administration may be preferred in someembodiments because of the convenience to the subject as well as thedosing schedule. Compositions suitable for oral administration may bepresented as discrete units such as hard or soft capsules, pills,cachettes, tablets, troches, or lozenges, each containing apredetermined amount of the active compound. Other oral compositionssuitable for use with the invention include solutions or suspensions inaqueous or non-aqueous liquids such as a syrup, an elixir, or anemulsion. In another set of embodiments, the composition may be used tofortify a food or a beverage.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or interperitoneal. The composition can be injectedinterdermally for treatment or prevention of infectious disease, forexample. In some embodiments, the injections can be given at multiplelocations. Implantation includes inserting implantable drug deliverysystems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. Inhalation includes administering thecomposition with an aerosol in an inhaler, either alone or attached to acarrier that can be absorbed. For systemic administration, it may bepreferred that the composition is encapsulated in liposomes.

In general, the compositions of the invention may be delivered using abioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene, polyvinylpyrrolidone, and polymers of lactic acidand glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid),poly(valeric acid), and poly(lactide-cocaprolactone), and naturalpolymers such as alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion.Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In certain embodiments of the invention, the administration of thecomposition of the invention may be designed so as to result insequential exposures to the composition over a certain time period, forexample, hours, days, weeks, months or years. This may be accomplished,for example, by repeated administrations of a composition of theinvention by one of the methods described above, or by a sustained orcontrolled release delivery system in which the composition is deliveredover a prolonged period without repeated administrations. Administrationof the composition using such a delivery system may be, for example, byoral dosage forms, bolus injections, transdermal patches or subcutaneousimplants. Maintaining a substantially constant concentration of thecomposition may be preferred in some cases.

Other delivery systems suitable for use with the present inventioninclude time-release, delayed release, sustained release, or controlledrelease delivery systems. Such systems may avoid repeatedadministrations in many cases, increasing convenience to the subject andthe physician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include, for example,polymer-based systems such as polylactic and/or polyglycolic acids,polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,polyorthoesters, polyhydroxybutyric acid, and/or combinations of these.Microcapsules of the foregoing polymers containing drugs are describedin, for example, U.S. Pat. No. 5,075,109. Other examples includenonpolymer systems that are lipid-based including sterols such ascholesterol, cholesterol esters, and fatty acids or neutral fats such asmono-, di- and triglycerides; hydrogel release systems; liposome-basedsystems; phospholipid based-systems; silastic systems; peptide basedsystems; wax coatings; compressed tablets using conventional binders andexcipients; or partially fused implants. Specific examples include, butare not limited to, erosional systems in which the composition iscontained in a form within a matrix (for example, as described in U.S.Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and5,239,660), or diffusional systems in which an active component controlsthe release rate (for example, as described in U.S. Pat. Nos. 3,832,253,3,854,480, 5,133,974 and 5,407,686). The formulation may be as, forexample, microspheres, hydrogels, polymeric reservoirs, cholesterolmatrices, or polymeric systems. In some embodiments, the system mayallow sustained or controlled release of the composition to occur, forexample, through control of the diffusion or erosion/degradation rate ofthe formulation containing the composition. In addition, a pump-basedhardware delivery system may be used to deliver one or more embodimentsof the invention.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments of the invention. “Long-term release,” as used herein, meansthat the implant containing the composition is constructed and arrangedto deliver therapeutically effective levels of the composition for atleast 30 or 45 days, and preferably at least 60 or 90 days, or evenlonger in some cases. Long-term release implants are well known to thoseof ordinary skill in the art, and include some of the release systemsdescribed above.

In some embodiments, the compositions of the invention may includepharmaceutically acceptable carriers with formulation ingredients suchas salts, carriers, buffering agents, emulsifiers, diluents, excipients,chelating agents, fillers, drying agents, antioxidants, antimicrobials,preservatives, binding agents, bulking agents, silicas, solubilizers, orstabilizers that may be used with the active compound. For example, ifthe formulation is a liquid, the carrier may be a solvent, partialsolvent, or non-solvent, and may be aqueous or organically based.Examples of suitable formulation ingredients include diluents such ascalcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate,or sodium phosphate; granulating and disintegrating agents such as cornstarch or algenic acid; binding agents such as starch, gelatin oracacia; lubricating agents such as magnesium stearate, stearic acid, ortalc; time-delay materials such as glycerol monostearate or glyceroldistearate; suspending agents such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone; dispersing or wetting agents such as lecithin orother naturally-occurring phosphatides; thickening agents such as cetylalcohol or beeswax; buffering agents such as acetic acid and saltsthereof, citric acid and salts thereof, boric acid and salts thereof, orphosphoric acid and salts thereof; or preservatives such as benzalkoniumchloride, chlorobutanol, parabens, or thimerosal. Suitable carrierconcentrations can be determined by those of ordinary skill in the art,using no more than routine experimentation. The compositions of theinvention may be formulated into preparations in solid, semi-solid,liquid or gaseous forms such as tablets, capsules, elixirs, powders,granules, ointments, solutions, depositories, inhalants or injectables.Those of ordinary skill in the art will know of other suitableformulation ingredients, or will be able to ascertain such, using onlyroutine experimentation.

Preparations include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,injectable organic esters such as ethyl oleate, or fixed oils includingsynthetic mono or di-glycerides. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, 1,3-butandiol, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents and inert gases and the like. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid may be used in the preparation of injectables.Carrier formulation suitable for oral, subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those of skillin the art can readily determine the various parameters for preparingand formulating the compositions of the invention without resort toundue experimentation.

In some embodiments, the present invention includes the step of forminga composition of the invention by bringing an active compound intoassociation or contact with a suitable carrier, which may constitute oneor more accessory ingredients. The final compositions may be prepared byany suitable technique, for example, by uniformly and intimatelybringing the composition into association with a liquid carrier, afinely divided solid carrier or both, optionally with one or moreformulation ingredients as previously described, and then, if necessary,shaping the product.

In some embodiments, the compositions of the present invention may bepresent as pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salts” includes salts of the composition, prepared incombination with, for example, acids or bases, depending on theparticular compounds found within the composition and the treatmentmodality desired. Pharmaceutically acceptable salts can be prepared asalkaline metal salts, such as lithium, sodium, or potassium salts; or asalkaline earth salts, such as beryllium, magnesium or calcium salts.Examples of suitable bases that may be used to form salts includeammonium, or mineral bases such as sodium hydroxide, lithium hydroxide,potassium hydroxide, calcium hydroxide, magnesium hydroxide, and thelike. Examples of suitable acids that may be used to form salts includeinorganic or mineral acids such as hydrochloric, hydrobromic,hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, phosphorous acids and the like. Other suitableacids include organic acids, for example, acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, glucuronic, galacturonic, salicylic, formic,naphthalene-2-sulfonic, and the like. Still other suitable acids includeamino acids such as arginate, aspartate, glutamate, and the like.

The invention also includes methods for quantitating a level ofprecursor microRNA expression. The method involves incorporating aprecursor microRNA into a reporter system, transfecting a host cell withthe reporter system, and detecting expression of a reporter gene productto quantitate the level of precursor microRNA expression. In someembodiments the reporter system includes a firefly luciferase reportergene.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

VI. Exemplary Uses

Drug Target Validation

Good drugs are potent and specific; that is, ideally, they must havestrong effects on a specific biological pathway or tissue (such as thedisease tissue), while having minimal effects on all other pathways orall other tissues (e.g., healthy tissues). Confirmation that a compoundinhibits the intended target (drug target validation) and theidentification of undesirable secondary effects are among the mainchallenges in developing new drugs.

Modern drug screening typically requires tremendous amounts of time andfinancial resources. Ideally, before even committing to such anextensive drug development program to identify a drug, one would like toknow whether the intended drug target would even make a good target fortreating a disease. That is, whether antagonizing the function of theintended target (such as a disease-associated oncogene or survivalgene), would be sufficient/effective to treat the disease, and whethersuch treatment would bear an acceptable risk or side effect. Forexample, if a cancer is determined to be caused by an activatingmutation in the Ras pathway, or caused by abnormal activity of asurvival gene such as Bcl-2, the subject system can be used to generateanimal models for drug target validation. Specifically, one can generatea transgenic mouse with the subject tet-responsive mishRNA expression,with the mishRNA targeting a gene that is a potential drug target (i.e.,Ras or Bcl-2 in this example). Tumors with various initiating lesionscan then be made in the mouse, and the mishRNA can be switched on in thetumor (if, for example, a tet-ON regulator is used). Such mishRNAexpression mimics the action of a (yet to be identified) drug that wouldinterfere with that target. If knocking down the target gene iseffective to reverse or stall the course of the disease, the target geneis a valid target.

Optionally, the mishRNA transgene can be switched on in a number oftissues or organs, or even in the whole organism, in order to verify thepotential side effects of the (yet to be identified) drug on otherhealthy tissues/organs.

Thus another aspect of the invention provides an animal useful for drugtarget validation, comprising a germline transgene encompassing thesubject artificial nucleic acid, which transcription is driven by a PolII promoter. The expression of the encoded precursor molecule (such asone based on the miR30-design) leads to an siRNA that targets acandidate drug target. Optionally, the precursor molecule is expressedin an inducible, reversible, and/or tissue-specific manner.

In a related aspect, the invention provides a method for drug targetvalidation, comprising antagonizing the function of a candidate drugtarget (gene) using a subject cell or animal (e.g., a transgenic animal)encompassing the subject artificial nucleic acid, either in vitro or invivo, and assessing the ability of the encoded precursor molecule toreverse or stall the disease progress or a particular phenotypeassociated with a pathological condition. Optionally, the method furthercomprises assessing any side effects of inhibiting the function of thetarget gene on one or more healthy organs/tissues.

Animal Disease Model

The subject nucleic acid constructs enables one to switch on or off atarget gene or certain target genes (e.g., by using crossing differentlines of transgenic animals to generate multiple-transgenic animals)inducibly, reversibly, and/or in a tissue-specific manner. This wouldfacilitate conditional knock-out or turning-on of any target gene(s) ina tissue-specific manner, and/or during a specific developmental stage(e.g., embryonic, fetal, neonatal, postnatal, adult, etc.). Animalsbearing such transgenes may be treated, such as by providing a tetanalog in drinking water, to turn on or off certain genes to allowcertain diseases to develop/manifest. Such system and methods areparticularly useful, for example, to analize the role of any known orsuspected tumor suppressor genes in the maintenance of immortalized ortransformed states, and in continued tumor growth in vivo.

In certain embodiments, the extent of gene knock-down may be controlledto achieve a desired level of gene expression. Such animals or cell(healthy or diseased) may be used to study disease progress, response tocertain treatment, and/or screening for drug leads.

The ability of the subject system to use a single genomic copy of thePol II promoter-driven mishRNA cassette to control gene expression isparticularly valuable for complex library screening.

The subject gene knock-down by expression of shRNA-mirs may be verysimilar to overexpression of protein-coding cDNAs. Thus any expressionsystems allowing targeted, regulated and tissue-specific expression,which have traditionally be limited to gene overexpression studies, maynow be adapted for loss-of-function studies, especially when combinedwith the available genome-wide, sequence-verified banks of miR-30-basedshRNAs targeting model organisms, such as human and mouse.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Introduction

In contrast to RNA Polymerase II promoters which are used by genesencoding proteins, RNA Polymerase III (Pol III) promoters, such as U6and H1 promoters, normally drive the transcription of several endogenoussmall nuclear RNAs (snRNAs). For this reason, Pol III promoters havebeen widely adopted to drive transcription of synthetic short hairpinRNAs (shRNAs) in cells and animals. Applicants and others have usedshRNAs driven by U6 promoters to achieve stable knockdown of targetgenes. Delivery of Pol III promoter-shRNA cassettes by retroviraltransduction of mammalian cells results in stable suppression of targetgene expression.

However, shRNA driven by Pol III promoters has certain practicalproblems. First of all, unlike Pol II promoters, the Pol III promotersdo not lend themselves to regulation. Secondly, such Pol III-drivenshRNAs can be ineffective inhibitors of their target mRNAs whenexpressed from a single-copy vector.

Here, Applicants have used certain RNA Polymerase II (Pol II) expressionsystems to allow potent and regulatable RNAi in mammalian cells.Applicants have shown that miR30 design shRNAs expressed from the LTRpromoter of an integrated retrovirus suppress target genes moreeffectively than when expressed from an RNA polymerase III promoter,even when expressed from a single-copy in the genome (e.g., from astably transfected or a transgenic copy). Furthermore, regulated shRNAexpression was also achieved by using inducible/reservible Pol IIpromoters, such as a Tet-responsive Pol II promoter.

Example I RNA Polymerase III Promoters are Sufficient for Stable shRNAExpression

In order to identify a preferred retroviral vector for delivery ofpromoter-shRNA cassettes into mammalian cells, Applicants compared twovectors based on the murine stem cell virus (MSCV) and theself-inactivating (SIN) retroviral vector, respectively. The 5′-longterminal repeat (5′-LTR) promoter of the SIN provirus istranscriptionally inactive, thus using the SIN 5′-UTR promoter in thisconstruct (e.g., USP-p53C, see below) serves as a control for theconstruct using the functional MSCV Pol II promoter (e.g., LUSP-p53C,see below).

Into each vector, Applicants cloned the U6 RNA polymerase III promoterupstream of a sequence encoding p53C, a short hairpin RNA (shRNA) thattargets murine p53. The shRNA p53C is predicted to fold into a simple,symmetrical shRNA with a 29-nucleotide stem and an eight nt loop (FIG.1A, left). Also in each vector, a puroR-IRES-GFP(PIG) cassette under thecontrol of the PGK promoter was operably linked downstream of theU6-shRNA cassette (FIG. 1B).

Primary murine embryonic fibroblasts (MEFs) were infected with eitherthe SIN-U6shRNA-PIG (UP)-p53C construct (USP-p53C), the MSCV-U6shRNA-PIG(LUP)-p53C construct (LUSP-p53C), or a control virus, and subject topuromycin selection to establish stably-infected cell lines.

After treatment with the DNA-damaging agent adriamycin to induce p53expression, cells stably-integrated by the above constructs wereharvested, and their p53 expression levels were assessed by Westernblotting. Interestingly, Applicants found that p53 knockdown was farmore effective in cells transduced with the SIN retrovirus (FIG. 1C),indicating that the internal U6 Pol III promoter is sufficient forexpression of the p53C shRNA in mammalian cells (since the SIN Pol IIpromoter is inactive in the USP-p53C construct). Also surprisingly, ourobservations suggest that transcription from the upstream MSCV LTRpromoter, a strong RNA polymerase II promoter, inhibited shRNA functionand p53 knockdown in this context. This effect may be due to promoterinterference between the LTR Pol II and U6 Pol III promoters.

Similar results were obtained for several other shRNAs with simplestem/loop folds similar to p53C (data not shown), verifying the generalapplicability of using RNA polymerase III promoters alone for expressionof this style (the simple stem-loop style) of shRNA.

Example II LTR Pol II Promoter is More Effective than RNA Polymerase IIIPromoter in Directing Integrated milR30-Design shRNAs Suppression ofTarget Genes

This example demonstrates that synthetic shRNAs with folds designed tomimic endogenous microRNA (miRNA) precursors can effectively inhibittarget gene expression. To illustrate, Applicants used the exemplarymiR30-design shRNAs (designated microRNA-based shRNAs, or mishRNAs) todemonstrate stable suppression of gene expression in mammalian cells,which strategy can be generally applied to other microRNA (miRNA)precursors. Specifically, Applicants recovered a mishRNA referred to asp53.1224 (so named because the predicted siRNA begins at nucleotide 1224of the p53 cDNA) from the mishRNA library (a genome wide miR30-basedshRNA library).

As shown above, standard stem-loop shRNAs are most effectively expressedfrom RNA polymerase III (Pol III) promoters such as the U6 promoter.Applicants sub-cloned a U6 promoter-p53.1224 cassette into a murine stemcell virus (MSCV) and a self-inactivating (SIN) retroviral vector, thusgenerating two vectors designed to express miR-based shRNAs (as opposedto the stem-loop shRNA): MSCV/LTR-U6miR30-PIG (LUMP)-p53.1224 andSIN-U6miR30-PIG (UMP)-p53.1224 (FIG. 1B). One difference between themishRNA and the standard stem-loop shRNA is that the miR30 precursor RNAis approximately 300 nt in length and is predicted to fold into anextensive secondary structure (FIG. 1A, right).

Applicants have previously constructed similar vectors expressing astandard stem-loop shRNA targeting p53 (p53C), producingMSCV/LTR-U6shRNA-PIG (LUSP)-p53C or SIN-U6shRNA-PIG (USP)-p53C (seeabove and FIG. 1B). All four constructs were introduced into earlypassage murine embryonic fibroblasts (MEFs). The resulting cellpopulations were assessed for p53 knockdown after adriamycin treatment(a DNA damaging agent that stabilizes p53), and the ability to formcolonies when plated at low density (a functional readout of p53 loss).

Contrast to what was observed for the simple stem-loop shRNA, theMSCV-based p53.1224 mishRNA (LUMP-p53.1224) driven by a functional PolII promoter was more effective at suppressing p53 than its S1N-basedcounterpart (UMP-p53.1224) devoid of a functional Pol II promoter,producing nearly undetectable p53 levels as assessed by immunoblotting(FIG. 1C, compare lanes 6, 7, and 8). As shown above, for the standardstem-loop shRNA, the SIN-based p53C shRNA (USP-p53C) was more effectiveat suppressing p53 than its MSCV-derived counterpart (LUSP-p53C),verifying that the U6 promoter is sufficient for stem/loop shRNAexpression (FIG. 1C). The ability of each vector to suppress p53correlated precisely with its ability to stimulate colony formation atlow density, with cells expressing the MSCV-based p53.1224 vectorproducing as many colonies as p53-null cells (FIG. 1D).

Southern blotting using a GFP probe verified that these differences werenot due to variation in retroviral copy number (data nor shown).

This vector preference was also observed for several other mishRNAs andstem-loop shRNAs targeting diverse gene products (data not shown). Thus,in general, mishRNAs can be remarkably potent when stably expressed fromretroviral vectors, particularly those with a functional 5′-LTR (with aPol II promoter). In the examples shown herein, this system achievednear-complete (if not complete) target gene knockdown.

Example III Pol II Promoter Contributes to Functional shRNA Production

The more potent knockdown produced by mishRNAs expressed from the MSCVvector compared with the SIN vector implies that the 5′-LTR contributesto optimal mishRNA expression. To determine whether the 5′-LTR promoter,a strong Pol II promoter, is sufficient for effective gene knockdownusing mishRNAs, Applicants introduced the p53.1224 shRNA into an MSCVvector lacking a U6 promoter (MSCV/LTRmiR30-PIG (LMP) (FIG. 1B). Tofacilitate comparison, Applicants introduced this vector and its LUMPand UMP counterparts into NIH3T3 cells at a low multiplicity ofinfection (<5% efficiency) such that the vast majority of transducedcells should contain single proviral integrations. Remarkably, bothvectors harboring the MSCV LTR (LUMP-p53.1224 and LMP-p53.1224)suppressed p53 expression extremely efficiently, and were far superiorto UMP-p53.1224, which expresses p53.1224 from the U6 promoter alone(FIG. 1E). Similar results were obtained in other cell types includingwild type and p19ARF-null MEFs (data not shown).

Thus, transcription of mishRNAs from Pol II promoters (such as theretroviral LTR in this example) is sufficient for highly effectivetarget gene knockdown, even when expressed at single copy, and even inthe absence of any Pol III promoters. Such features are extremelyvaluable for knockdown screens using complex libraries, where infectedcells are unlikely to contain multiple copies of a given shRNA vector.

The fact that the 5′-LTR Pol II promoter produced results similar tothose of the 5′-LTR+U6 promoters (Pol II and Pol III promoters),suggested that in this case, U6 may be mainly acting as a “spacer.” Asthe combined effects of the 5′-LTR and U6 promoters appeared to be moreeffective than U6 alone, promoter interference is unlikely, and rather,it suggests dominance of the LTR promoter.

Interestingly, GFP is less abundant in cells with LTR-miR30 transcript.While not wishing to be bound by any particular theory, this is likelydue to degradation of this transcript after nuclear processing byDrosha. Since microRNA clusters, much GFP appeared to be translated fromthe IRES on the long LTR transcript.

Example IV in vivo Loss-of-Function Phenotypes can be RecapitulatedUsing miR30-Design shRNAs Expressed from Pol II Promoters

Stable expression of stem/loop shRNAs can produce loss of functionphenotypes in mice. To determine whether miR30-derived shRNAs expressedfrom pol II promoters can efficiently modulate gene expression in vivo,Applicants targeted genes for which the null phenotype was known. Forexample, inactivation of the BH3-only protein Bim (a pro-apoptoticmember of the Bcl-2 family) accelerates lymphomagenesis in Eμ-myctransgenic mice. To this end, Applicants have demonstrated thatmiR30-design shRNAs targeting Bim would also cooperate with myc duringlymphomagenesis. Indeed, mice reconstituted with Eμ-myc hematopoeticstem cells (HSCs) transduced with two independent miR30-design shRNAstargeting Bim (collectively referred to as shBim, and expressed from theLTR of MSCV/LTRmiR30-SV40-GFP (LMS), a derivative of LMP that lacks aPol III promoter) formed tumors much more rapidly than animalsreconstituted with stem cells expressing a control vector (FIG. 2A,P<0.05). Importantly, lymphomas arising in animals transduced with shBimwere GFP-positive, expressed low levels of Bim (FIG. 2B), and displayeda mature (IgM⁺) B cell phenotype uniquely characteristic of Bim nulllymphomas (data not shown). Thus, in vivo loss of function phenotypescan be recapitulated using miR30-design shRNAs expressed from Pol IIpromoters.

Example V Identification and Characterization of Genes that Modify DrugResponses in vivo

siRNAs have been used to identify modulators of drug action, but are notsuitable for long-term assays or in vivo systems. The miR30-basedvectors described above enable the identification and characterizationof genes that modify drug responses in vivo.

As an illustrative example, Applicants examined the ability of amiR30-design p53 shRNA to promote chemoresistance in Eμ-myc lymphomas,which respond poorly to therapy in the absence of p53. Applicantsintroduced LMS-p53.1224 (co-expressing GFP) into chemosensitive Eμ-myclymphoma cells at ˜10% infection efficiency and transplanted the mixedcell populations into syngeneic recipient mice. Upon lymphomamanifestation, animals were treated with the chemotherapeutic drugadriamycin and monitored for tumor response. Strikingly, mice harboringlymphomas expressing LMS-p53.1224 did not regress following adriamycintreatment and showed significantly reduced overall survival relative tocontrol tumor-bearing mice (FIG. 2C). This indicates that theLMS-p53.1224 construct effectively knocked-down p53 expression in tumorcells, resulting in their poor response (or chemoresistance) to therapy.Furthermore, the percentage of GFP positive cells dramatically increasedin lymphomas harboring p53.1224 but not the control vector, indicating aselective advantage for p53.1224 expressing cells (FIG. 2D).

These results demonstrated that sufficient p53 knockdown may promote invivo chemoresistance. Such an animal mode (or tumor cells therein) mayalso be used to screen (in vivo or in vitro) for compounds that canovercome chemoresistance in p53 negative cells.

Together, these data indicate that mishRNAs expressed from Pol IIpromoters are suitable for a variety of in vivo applications, withstrong potential for transgenic animals, tissue specific gene knockdownsand in vivo forward genetic screens.

Example VI Pol II Promoter-Driven Inducible and Reversible shRNAProduction from Low-Copy Stable Integration

RNAi inhibits gene function without altering DNA sequence, therefore itseffects are potentially reversible. Given our findings that low copy PolII promoters can effectively drive mishRNAs from a single integratedconstruct, Applicants adapted the traditional inducible proteinexpression systems, such as the tetracycline (tet)-regulated Pol IIpromoter TRE-CMV, to achieve inducible stable expression of mishRNAs.

Many inducible promoters are known in the art in the context of proteinexpression. These inducible systems can all be adpated to express themishRNAs of the subject invention. In one illustrative example, theTRE-CMV promoter consists of a tandem array of tet transactivatorbinding sites fused to a minimal CMV promoter. Transactivator proteintTA transactivates the TRE-CMV promoter in the absence of thetetracycline analog doxycycline (Dox). This promoter system has beenshown to be highly effective for conditional expression ofprotein-coding cDNAs both in vitro and in vivo. Thus when adapted foruse in the subject invention, shRNA expression can mediate target geneknockdown in the absence of Dox both in vitro and in vivo.

Using a SIN vector backbone, Applicants cloned a mishRNA targeting humanRb (Rb.670) downstream of the TRE-CMV promoter, producingSIN-TREmiR30-PIG, or TMP-Rb.670; FIG. 3A). HeLa cells stably expressingthe tet transactivator protein tTA (tet-off) were infected withTMP-Rb.670 at single copy in the absence of Dox. Rb levels in these cellpopulations were slightly decreased compared with uninfected controls,indicating potential shRNA production from the TRE-CMV promoter (FIG.3B). Indeed, when single cell clones were generated from thispopulation, 6 of 13 showed excellent Rb knockdown (FIG. 3C and data notshown), demonstrating that the TRE-CMV promoter can effectively driveshRNA expression at low copy number.

To examine inducible regulation of shRNA expression, Rb.670C cells,which showed significant Rb knockdown in Dox-free medium (FIG. 3C), werecultured in various Dox concentrations for many days. Cell growth andviability were indistinguishable at all Dox concentrations. However,Applicants observed a clear dose-dependency of Rb expression, withmaximum Rb knockdown achieved in low Dox concentrations and vice versa(FIG. 3D). At Dox concentrations of less than 0.005 ng/mL, Dox producedminimal Rb expression. However, cells grown in 0.008 ng/mL Dox showedslight de-repression of Rb. Normal Rb expression was restored in cellscultured in approximately 0.05 ng/mL Dox and higher, suggesting thatshRNA expression is not leaky at these Dox concentrations.

Thus, Dox concentration can tightly control the extent of stable geneknockdown. This effect was also observed in time-course studies, whereApplicants observed normalization of Rb expression upon Dox addition,and rapid Rb knockdown upon Dox removal (FIG. 3E), demonstrating thereversibility of the induced mishRNA expression. Remarkably, in allcases GFP and Rb levels were inversely correlated (FIGS. 3D and 3E),with intermediate GFP expression observed between 0.002 and 0.008 ng/mLDox. As GFP and shRNA are produced from the same transcript, GFPexpression may be regarded as a surrogate marker of shRNA production.

A great advantage of tet-regulated systems is that expression from theTRE-CMV promoter can be either induced (tet-ON) or repressed (tet-OFF)by Dox, depending on which tet transactivator protein is used. To test atet-ON shRNA expression system, Applicants utilized U2OS cells stablyexpressing the reverse tTA (rtTA) protein, which in contrast to tTA,requires Dox to activate transcription.

Applicants also isolated a clone (Rb.670R5; FIG. 3F) of U2OS cellsinfected with TMP-Rb.670 and stably expressing the reverse tTA (rtTA;tet-on) protein. As predicted, Dox concentration and Rb knockdown werepositively correlated in these cells (FIGS. 3G and 3H).

At Dox concentrations of less than 0.005 ng/mL, Dox produced minimal Rbexpression. However, cells grown in 0.008 ng/mL Dox showed slightde-repression of Rb. Normal Rb expression was restored in cells culturedin approximately 0.05 ng/mL Dox and higher, suggesting that shRNAexpression is not leaky at these Dox concentrations. As GFP protein istranslated from an IRES, it can be produced from transcripts originatingfrom both the PGK and TRE-CMV promoters. As GFP is not detected in cellsgrown in high Dox concentrations, it appears that GFP production fromthe PGK promoter transcript is very weak. Our results suggest that themajority of GFP in untreated Rb.670C cells arises from the CMV-TREtranscript, production of which is blocked by Dox in a dose-dependentmanner. As the TRE-CMV transcript also carries the miR30-based shRNAfold, GFP expression may be regarded as a surrogate marker of shRNAproduction.

Using the same Tet-responsive system, good protein expression regulationwas also achieved in several other cell clones, including thoseexpressing a PTEN-miR30 construct.

These observation verifies that low copy delivery of the TMP vector(also lacking a Pol III promoter) allows regulated mishRNA expression ineither tet-on or tet-off configurations, and altering Dox concentrationin this system allows tight control of the extent of stable geneknockdown.

Example VII Reversible Induction of Pol II-Driven Tet-Responsive p53shRNA Production in Primary Cells

The instant regulatable shRNA expression is not only operable inimmortalized cell lines, but also functional to regulate suppression ofgenes (e.g., the tumor suppressor gene p53) in primary cells.

For example, inactivation of the tumor suppressor p53 immortalizes wildtype MEFs, and transforms MEFs over-expressing oncogenic Ras. Earlypassage MEFs were co-transduced with TMP-p53.1224 and a retrovirusexpressing the tTA (tet-off) protein. Many doubly infected MEFs(designated wild type/tTA/TMP-p53.1224, or WtT) showed stable p53knockdown when cultured in Dox-free medium. WtT cells plated at lowdensity formed colonies comparable in size and number to those formed byp53-null MEFs (FIG. 4A), suggesting that p53 was functionallyinactivated in most cells. Colony formation of WtT cells cultured in Doxin parallel was similar to that of control cells (FIG. 4A), suggestingnormal p53 expression. p53-null MEF growth was unaffected by Dox, rulingout non-specific effects (FIG. 4A).

Applicants also isolated several WtT clones and examined their p53regulation in response to Dox. p53 expression in WtT cells increasedrapidly and GFP expression was lost upon Dox treatment (FIGS. 4B and4C). WtT clones cultured in Dox failed to form colonies when plated atlow density. Instead, by day 8 of Dox treatment, all cells had aflattened morphology characteristic of senescent cells, and many werepositive for senescence-associated β-galactosidase (SA-β-gal; FIG. 4C).This dormant phenotype was stable for weeks of continuous culture inDox. Therefore, p53.1224 shRNA expression can be tightly regulated byDox treatment in wild type MEFs doubly infected with tTA andTMP-p53.1224.

The rapid and coordinated senescence response observed when endogenousp53 expression was restored in MEFs immortalized by p53 knockdown wasreversed upon Dox removal (FIG. 4D, left panel, upper well), inagreement with previous observations in other MEF culture systems.Control cells continually cultured in Dox remained dormant (FIG. 4D,left panel, lower well). Newly proliferating cells (FIG. 4D, left panel,upper well) remained responsive to p53 re-expression, as they lost GFPexpression and failed to form colonies when re-plated in Dox (FIG. 4D,right panel, lower well).

These results suggest that wild type MEFs can be reversibly switchedbetween cycling and senescent states simply by regulating p53 knockdown.WtT cells transformed by infection with activated Ras (FIG. 4E, upperpanels) also became morphologically senescent and SA-β-gal positive whentreated with Dox (FIG. 4E, lower panels), with p53 and GFP expressionchanges similar to that of parental WtT cells (FIG. 4F).

Furthermore, Applicants conclude that restoration of p53 expression inwild type MEFs immortalized by stable p53 knockdown causes a rapid andcoordinated senescence response. This demonstrates that at least incancers with p53 loss-of-function mutations, cancers can be treated byrestoring p53 expression to induce senescence. This technique can alsobe extended to test any potential target genes whose functions are lostin diseases, such as in cancer. Specifically, the system of the instantinvention may be used to test whether loss-of-function of a candidategene causes certain disease state, and whether restoring such targetgene function in diseased tissues can reverse the disease status, or atleast slow down disease progression.

Example VIII Reversible in vivo Gene Knockdown Using Tet-ResponsivePromoter

Tet-regulated over-expression systems have revolutionized the study ofthe role of oncogenes in tumor survival in vivo. Tet-regulated RNAiholds similar promise for regulated knockdown of tumor suppressor genes.To illustrate this concept, Applicants injected WtT-Ras MEFssubcutaneously into the flanks of nude mice formed visible, rapidlygrowing and strongly GFP positive tumors after approximately 2 weeks,verifying that these cells were functionally transformed (FIG. 5A; upperpanels). To inactivate p53.1224 shRNA in established tumors, mice weretreated with Dox via their drinking water. After only 2 days of Doxtreatment, tumor GFP intensity was markedly diminished compared withuntreated mice, and after 4 days tumors were almost GFP negative (FIG.5A). Remarkably, tumor growth slowed upon Dox treatment, and tumorsbegan shrinking after approximately 4 to 6 days (FIG. 5B). Animalstreated with Dox for 10 days often showed continued tumor regression andbecame tumor-free (FIG. 5B). This regression was p53-dependent, astumors derived from p53-null MEFs infected with tTA, TMP-53.1224 and Raslost GFP expression but continued to grow when treated with Dox (datanot shown). Similar results were obtained for several WtT-Ras clones andWtT clones infected with EIA/Ras, with variable tumor growth rates andregression kinetics (data not shown).

Dox-treated animals with regressing tumors were taken off Dox treatmentafter various times. In many cases, usually when initial tumor size wasless, mice became tumor-free and remained so for weeks. However,removing Dox from animals with larger regressing tumors or after abriefer Dox treatment often allowed renewed GFP expression and tumorgrowth (FIG. 5C). Interestingly, tumors isolated from Dox-treatedanimals contained cells with unusually compact nuclei, and widespreadapoptosis was seen compared with untreated controls (FIG. 5D),suggesting that tumor regression was at least in part due top53-dependent apoptosis. Indeed, as predicted, p53 expression wasdramatically elevated in tumors from Dox-treated animals (FIG. 5E).

In summary, by adapting a standard Pol II promoter-driven tet-responsivepromoter normally used for inducible protein expression, Applicants forthe first time have demonstrated inducible and reversible target geneknockdown in vivo. p53 re-expression in tumors caused regressionassociated with widespread apoptosis, in contrast to the senescenceobserved when p53 was re-expressed in the same cells in culture. Thesefindings highlight the ability of this technology for the study of manyaspects of biology, including identification and/or validation ofpotential drug targets in animal models. The tet system has obviousadvantages over unidirectional Cre-lox strategies, and many keyreagents, such as tissue-specific tet transactivator mice, are readilyavailable.

In summary, expression of miRNA-design short hairpin RNAs (shRNAs)allows stable, post-transcriptional suppression of gene activity, whichis optionally reversible. Applicants have developed a new retroviralvector system that uses RNA polymerase II promoters to express shRNAsbased on the human miR30 precursor. Single copy expression of shRNAsfrom this vector yields potent and stable gene knockdown in culturedcells and in vivo. Expression of an shRNA targeting p53 using thissystem mimics complete p53 loss and renders tumor cells chemoresistantin vivo. By improving standard tet-inducible promoters for shRNAexpression, we show stable, incremental, and reversible gene knockdownof various target genes in tet-on or tet-off configurations.Interestingly, cultured wild type mouse fibroblasts can be switched fromproliferative to senescent states simply through regulated knockdown ofp53. We find that tumors derived from wild type mouse fibroblaststransformed by Ras overexpression and p53 knockdown regress upon p53re-activation in vivo, suggesting that ongoing suppression of p53 isessential for tumor maintenance in this context. This system provesuseful for studying potential therapeutic targets in cancer, and in mostother biological systems.

All vectors described in these experiments are compatible withgenome-wide, sequence verified banks of miR30 shRNAs (or any othersimilar banks of miR shRNAs) targeting human and mouse genes, creating aformidable resource for diverse, large scale RNAi studies in mammaliansystems.

Methods

The following methods and reagents were used in the Examples above.These are merely for illustrative purpose, and are by no means limiting.Other comparable minor variations can be readily made without undueexperimentation for adapting to specific problems.

Vector Construction.

The retroviral vector MSCV-PIG has an EcoRI site in the polylinker andanother between the Puro® cassette and the IRES sequence. To facilitatecloning into the polylinker, the second site was destroyed using aPCR-based strategy: a PCR product was generated using MSCV-PIG template,forward primer 5′-TCTAGGCGCCGGAATTAGATCTCTCG-3′ (SEQ ID NO: 1), andreverse primer 5′-CCTGCAATTGGATGCATGGGGTCGTGC-3′ (SEQ ID NO: 2), anddigested with BgIII and MfeI. This fragment was cloned into MSCV-PIGdigested with BgIII/EcoRI, yielding MSCV-PIGdRI. MSCV-U6miR30-PIG wasmade by ligating the 762 bp BamHI-MfeI “U6-miR30 context” cassette frompSM2 into MSCV-PIGdRI digested with BgIII/EcoRI. MSCV-LTRmiR30-PIG wasmade by ligating the 256 bp SaII-MfeI “miR30 context” cassette from pSM2into MSCV-PIGdRI digested with XhoI/EcoRI. MSCV-LTRmiR30—SV40GFP (LMS)was made in two steps. Firstly, a ˜1.2 kb EcoRI-ClaI SV40GFP fragmentfrom pBabeGFP was ligated into MSCV-puro (Clontech) digested withEcoRI/ClaI, forming MSCV-SV40GFP. This was digested with XhoI/EcoRI, andthe 256 bp SaII-MfeI “miR30 context” cassette from pSM2 was inserted,forming MSCV-LTRmiR30-SV40GFP. SIN-PIGdRI was made by ligating the 2524bp EcoRI-SaII fragment from MSCV-PIGdRI into pQCXIX (Clontech) digestedwith EcoRI/XhoI. SIN-TREmiR30-PIG was constructed in two steps. Firstly,a PCR product spanning the TRE-CMV promoter was generated using templateplasmid pQTXIX (a kind gift from Abba Malina, generated by cloning theXbaI-EcoRI TRE-CMV promoter fragment from pUHD10.3 into pQCXIX(Clontech) digested with XbaI/EcoRI), using the primers 5′-GAATTGAAGATCTGGGGGATCGATC-3′ (SEQ ID NO: 3) and 5′-CATCAATTGCTAGAATTCTGGTTGCTCGAGAGGCTGGATCGGTCCCGGTGTCTTC-3′ (SEQ ID NO:4). This PCR product wasdigested with BgIII/MfeI and ligated into SIN-PIGdRI digested withBgIII/EcoRI (removing its CMV promoter), forming SIN-TRE-PIG.SIN-TREmiR30-PIG was completed by ligating the 256 bp SaII-MfeI “miR30context” cassette from pSM2 into SIN-TRE-PIG digested with XhoI/EcoRI.DNA fragments encoding various mishRNA folds were generated usingoligonucleotide template PCR as described previously, or subcloned as110 bp XhoI/EcoRI fragments from the pSM2 mishRNA library.Oligonucleotides were designed atkatahdin.cshl.org:9331/siRNA/RNAi.cgi?type=shRNA (incorporated herein byreference). PCR products were digested with XhoI/EcoRI and ligated intothe unique XhoI/EcoRI sites within the “miR30 context” in the vectorsdescribed above.

Cell Culture and Expression Analysis

Cells were grown in DMEM containing 10% fetal bovine serum at 37° C.with 7.5% CO₂. Doxycycline (Clontech) was dissolved in water andgenerally used at a final concentration of 100 ng/mL. Medium containingDox was refreshed every two days. Infections and colony formation assayswere carried out as previously described. SA-β-gal activity was detectedas previously described, with sample equilibration and X-gal stainingdone at pH 5.5. For western blotting analysis, Laemmli buffer proteinlysates were run on SDS-PAGE, and transferred to Immobilon PVDF membrane(Millipore). Antibodies were anti-p53 (1:1000 IMX25, VectorLaboratories), anti-PTEN (1:1000 486, a kind gift from Michael Myers),anti-GFP (1:5000, Clontech), anti-tubulin (1:5000 B-5-1-2, Sigma),anti-actin (1:5000, Sigma), and anti-Rb (1:1000 G3-245, Pharmingen with1:100 XZ-55 and C36 hybridoma supernatants).

Lymphoma Studies.

Eμ-myc lymphomagenesis and drug treatment studies were performed aspreviously described (Schmitt, 2000; Hemann, 2003). Chemosensitivelymphoma cells were isolated from tumors arising in mice transplantedwith Eμ-myc; P19^(ARF)+/−HSCs, which invariably lose the wild typep19^(ARF) allele while retaining wild type p53.

Nude Mouse Studies

Approximately 10⁶ transformed cells were injected subcutaneously intothe two rear flanks of nude mice. Mice were treated with 0.2 mg/mL Doxin a 0.5% sucrose solution in light-proof bottles, refreshed every fourdays. Tumor volume (mm³) was calculated as (length×width²×π/6). Foranalysis of protein expression, tumors were snap-frozen and pulverisedin liquid nitrogen using a mortar and pestle. Powdered tumor was lysedin Laemmli buffer and western analysis was performed as above. Forhistology, tumor tissue was fixed for 24 hours in 4% formaldehyde in PBSprior to embedding and sectioning. Apoptosis was measured by TUNEL assay(In situ Cell Death Detection Kit, POD; Roche).

Results described herein above are published in Nat. Genet. 37(11):1289-95, 2005 (Dickins et al., 2005). Other related work is published inNat. Genet. 37(11): 1281-88, 2005 (Silva et al., 2005). The entirecontents of these publications, including the related online(supplemental) information and contents of the publications citedtherein are incorporated herein by reference. The subject system canalso be used in lentiviral, pre-miR-30 based siRNA expression vectors,such as those including a tetracyclin-responsive Pol II promoter andthus can be used to tightly regulate the expression of target genes intransduced cells. See Stegmeier et al., Proc. Natl. Acad. Sci. U.S.A.102: 13212-17, 2005 (incorporated herein in its entirety).

Example IX In vivo Transgenic Animal Model for Tissue-Specific andInducible Target Gene Knockdown

This example demonstrates knockdown of a target gene, e.g., p53, in atissue-specific, inducible and/or reversible manner, in a germline(transgenic) animal model.

To achieve regulated transgene expression in germline transgenic mice,two lines of transgenic mice were generated: one expressing a tettransactivator protein (either tTA/tet-off or rtTA/tet-on), optionallyin a tissue-specific manner using tissue-specific promoters; and anotherharboring a tetracycline-responsive (TRE) promoter driving the transgeneof interest. Crossing these two lines yielded double transgenic micethat expressed the transgene, in a Dox-regulatable manner (either tet-onor tet-off), in cells where the transactivator (tTA or rtTA) wasexpressed.

Alternatively, the tTA or rtTA construct may be introduced (e.g. viainfection or transfection, etc.) into cells of a transgenic animalbearing TRE-mishRNA-expression cassette.

For example, to demonstrate that tet-regulated miR30-based shRNAexpression can be achieved in animals, Applicants generated severaltransgenic founder lines harboring a TRE-p53.1224 shRNA cassette (usingstandard pronuclear injection protocols). To test shRNA activity inthese animals, Applicants isolated MEFs (mouse embryonic fibroblasts)from F2 transgenic embryos and wild-type controls, infected them with aretrovirus expressing the tTA (tet-off) protein, and assessed p53knockdown after p53 induction by adriamycin. Specifically, primary MEFsderived from embryos from a cross between wild-type B6 females mated toTRE-p53.1224 founder lines A and B, were infected with eithertTA-IRES-Neo or tTA-IRES-GFP retrovirus. Then tTA-IRES-Neo MEFs wereselected for 7 days in G418 prior to harvesting in order to eliminateuninfected cells. The tTA-IRES-GFP MEFs were unselected, though the MEFswere infected at high percentage. All cells were adriamycin treatedprior to harvesting.

Of the two founder lines tested so far, one (line A) showed striking p53knockdown in transgene-positive cells (results not shown). Thisknockdown was similar to that seen when the p53.1224 shRNA was expressedfrom a retroviral LTR promoter (supra; Dickins et al., Nat. Genet.37(11): 1289-95, 2005). Importantly, p53 induction was normal inuninfected transgene-positive cells (e.g., by comparing founder line AMEFs either uninfected or infected with tTA-IRES-Neo. All cells wereadriamycin treated prior to harvesting. Results not shown). Thisdemonstrates that shRNA expression and p53 knockdown is tTA-dependentand not leaky.

Moreover, as expected, these MEF lines showed a rapid re-expression ofp53 upon Doxycycline treatment, indicating that shRNA expression wastightly controlled by Doxycycline (results not shown).

To our knowledge, the above experiments for the first time demonstratedthat tetracycline effectively regulated shRNA expression in a germlinetransgenic setting. This enables one to reversibly switch any endogenousgene on or off, simply by administering a reversible activator orinhibitor of a transcriptional regulator, such as Doxycycline (or otherTet homolog), preferably via drinking water. This technology isespecially powerful in examining gene function in vivo, for example,during embryonic or postnatal development, tumorigenesis, or aftertreatment of tumors with chemotherapeutic drugs.

As indicated above, Applicants have also crossed the TRE-p53.1224transgenic mice to established transgenic lines that express the tTA(tet-off) protein in a tissue-specific manner. As expected, Applicantsdetected p53.1224 siRNA in the liver of LAP-tTA; TRE-p53.1224 doubletransgenic mice, where tTA expression was restricted to the liver (lane2 of FIG. 6). After 4 days of doxycycline administration, someattenuation of siRNA production was observed (lane 3 of FIG. 6).Applicants have also been assessing p53 knockdown in the liver of thesemice, in order to determine whether longer term doxycyclineadministration will further or completely block siRNA production. Notethat the spleens of these mice were devoid of siRNA (see lanes 4-6 ofFIG. 6), consistent with liver-specific expression of the siRNA.

The system can also be used to generate animal models for studying theeffect of turning on/off certain target genes in the progression ofcertain diseases, such as cancer.

For example, the Eμ-myc mouse is prone to developing lymphoma, which isaccelerated further by loss of p53 function. To model this process usingtet-regulated p53 knockdown in vivo, Applicants crossed Eμ-myc mice toTRE-p53.1224 mice and Eμ-tTA mice, which expressed tTA specifically inthe B cell compartment. As myc and tTA should be expressed coordinatelyin B cells of the Eμ-myc; Eμ-tTA; TRE-p53.1224 triple transgenic mice,Applicants expected reversible p53 knockdown in oncogene-expressingcells.

Consistent with this prediction, in a spleen (a tissue enriched forlymphoma cells) isolated from lymphoma-laden triple transgenic mice,Applicants observed highly abundant p53.1224 siRNA, at levels known topromote p53 knockdown and tumor progression (lanes 9-11 of FIG. 6). Incontrast, spleens from Eμ-myc; TRE-1224 double transgenic mice do notexpress the siRNA, indicating that the TRE-1224 transgene requires tTAfor expression.

Lymphoma cells isolated from these triple transgenic mice were thentransplanted into several recipient nude mice to allow controlled p53re-activation. Specifically, tumor-bearing recipient mice were treatedwith Doxycycline. Survival of heavily tumor-bearing transplantrecipients was extended by many days when doxycycline was administeredvia the drinking water. Furthermore, p53.1224 siRNA expression wascompletely suppressed in the lymph nodes and spleen of these treatedmice, indicating effective switching of shRNA expression in vivo.

These results demonstrated that Applicants can produce transgenic micewhere miR30-based shRNA production was tissue-specific, and can beinducibly and reversibly regulated simply by administering or omittingdoxycycline in the drinking water.

The practice of aspects of the present invention may employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).All patents, patent applications and references cited herein areincorporated in their entirety by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An artificial nucleic acid construct comprising an RNA Polymerase II(Pol II) promoter operably linked to a coding sequence for expressing aprecursor molecule for an siRNA, said siRNA inhibiting the expression ofa target gene, wherein the nucleic acid construct directs the expressionof the precursor molecule and/or the siRNA, and substantially inhibitsthe expression of the target gene when stably integrated into a hostcell genome.
 2. The nucleic acid construct of claim 1, wherein said PolII promoter is an inducible promoter, a tissue-specific promoter, and/ora developmental stage-specific promoter.
 3. The nucleic acid of claim 2,wherein the inducible promoter is a tetracyclin-responsive promoter. 4.The nucleic acid construct of claim 3, wherein thetetracyclin-responsive promoter is a TetON promoter, the transcriptionfrom which promoter is activated at the presence of tetracyclin (tet),doxycycline (Dox), or a tet analog.
 5. The nucleic acid construct ofclaim 3, wherein the tetracyclin-responsive promoter is a TetOFFpromoter, the transcription from which promoter is turned off at thepresence of tetracyclin (tet), doxycycline (Dox), or a tet analog. 6.The nucleic acid construct of claim 2, wherein the Pol II promoter is anLTR promoter or a CMV promoter.
 7. The nucleic acid construct of claim2, wherein the precursor molecule is a precursor microRNA.
 8. Thenucleic acid construct of claim 7, wherein the precursor microRNA (miR)is an artificial miR comprising coding sequence for said siRNA for saidtarget gene.
 9. The nucleic acid construct of claim 8, wherein the miRcomprises a backbone design of microRNA-30 (miR-30).
 10. The nucleicacid construct of claim 8, wherein the miR comprises a backbone designof miR-15a, -16, -19b, -20, -23a, -27b, -29a, -30b, -30c, -104, -132s,-181, -191, -223.
 11. The nucleic acid construct of claim 2, wherein theprecursor molecule is a short hairpin RNA (shRNA).
 12. The nucleic acidconstruct of claim 1, wherein a single integrated copy of the nucleicacid construct is sufficient for substantially inhibiting the expressionof the target gene.
 13. The nucleic acid construct of claim 1, furthercomprising an enhancer for the Pol II promoter.
 14. The nucleic acidconstruct of claim 1, further comprising a reporter gene under thecontrol of a second promoter.
 15. The nucleic acid construct of claim14, wherein the second promoter and the reporter gene is downstream of(3′-to) the coding sequence for the precursor molecule.
 16. The nucleicacid construct of claim 15, wherein the reporter gene is translated froman internal ribosomal entry site (IRES) between a second promoter andthe reporter gene.
 17. The nucleic acid construct of claim 1, furthercomprising at least one selectable marker.
 18. The nucleic acidconstruct of claim 1, further comprising a reporter gene, wherein thecoding sequence for expressing the precursor molecule is embedded orinserted into the 5′-UTR (untranslated region), 3′-UTR, or an intron ofthe reporter gene.
 19. The nucleic acid construct of claim 1, furthercomprising a Pol III promoter upstream of the coding sequence forexpressing the precursor molecule.
 20. The nucleic acid construct ofclaim 1, wherein the target gene is associated with a disease conditionselected from cancer or infectious disease.
 21. The nucleic acidconstruct of claim 20, wherein the target gene is over-expressed orabnormally active in the disease.
 22. The nucleic acid construct ofclaim 20, wherein the target gene is an oncogene or anantagonist/inhibitor or dominant negative mutation of a tumor suppressorgene.
 23. A cell comprising the nucleic acid construct of claim
 1. 24.The cell of claim 23, which is a mammalian cell.
 25. The cell of claim23, wherein the Pol II promoter is an inducible promoter, and whereinthe cell further comprises an additional construct for expressing anactivator or an inhibitor of the inducible promoter.
 26. The cell ofclaim 25, wherein the inducible promoter is a tet-responsive promoter,and wherein the additional construct encodes tTA or rtTA.
 27. Anon-human mammal comprising the cell according to claim
 23. 28. Thenon-human mammal of claim 27, which is a chimeric mammal.
 29. Thenon-human mammal of claim 27, which is a transgenic mammal.
 30. A methodfor inhibiting the expression of a target gene of interest in a cell,comprising introducing a construct according to claim 1 into the cell,wherein the siRNA molecule derived from the precursor molecule isspecific for the target gene.
 31. The method of claim 30, furthercomprising inhibiting at least one additional target gene(s) of interestin the cell by introducing at least one additional constructs accordingto claim 1 into the cell, wherein each of the siRNA molecules derivedfrom the precursor molecules are specific for the additional targetgenes, respectively.
 32. A method for treating a gene-mediated disease,comprising introducing into an individual having the disease a constructaccording to claim 1, where the siRNA derived from the precursormolecule is specific for the gene mediating the disease.
 33. A method ofvalidating a gene as a potential target for treating a disease,comprising: (1) introducing a construct according to claim 1 into a cellassociated with the disease, wherein the siRNA molecule derived from theprecursor molecule is specific for the gene; (2) assessing the effect ofinhibiting the expression of the gene on one or more disease-associatedphenotype; wherein a positive effect on at least one disease-associatedphenotype is indicative that the gene is a potential target for treatingthe disease.